Antibodies to insulin-like growth factor i receptor

ABSTRACT

The present invention relates to antibodies and antigen-binding portions thereof that specifically bind to insulin-like growth factor I receptor (IGF-IR), which is preferably human IGF-IR. The invention also relates to human anti-IGF-IR antibodies, including chimeric, bispecific, derivatized, single chain antibodies or portions of fusion proteins. The invention also relates to isolated heavy and light chain immunoglobulin molecules derived from anti-IGF-IR antibodies and nucleic acid molecules encoding such molecules. The present invention also relates to methods of making anti-IGF-IR antibodies, pharmaceutical compositions comprising these antibodies and methods of using the antibodies and compositions thereof for diagnosis and treatment. The invention also provides gene therapy methods using nucleic acid molecules encoding the heavy and/or light immunoglobulin molecules that comprise the human anti-IGF-IR antibodies. The invention also relates to gene therapy methods and transgenic animals comprising nucleic acid molecules of the present invention.

The present application is a continuation application of U.S. patentapplication Ser. No. 13/169,474, filed Jun. 27, 2011, which is acontinuation application of U.S. patent application Ser. No. 12/756,417,filed Apr. 8, 2010 and issued as U.S. Pat. No. 7,982,024 on Jul. 19,2011, which is a divisional application of U.S. patent application Ser.No. 11/144,248, filed Jun. 2, 2005 and issued as U.S. Pat. No. 7,815,907on Oct. 19, 2010, which is a continuation application of U.S. patentapplication Ser. No. 10/038,591, filed Jan. 4, 2002 and issued as U.S.Pat. No. 7,037,498 on May 2, 2006, which claims the benefit of U.S.Provisional Application No. 60/259,927, filed Jan. 5, 2001. Thedisclosures, including the claims, of all the aforementioned priorityapplications are incorporated by reference in their entirety herein.

BACKGROUND OF THE INVENTION

Insulin-like growth factor (IGF-I) is a 7.5-kD polypeptide thatcirculates in plasma in high concentrations and is detectable in mosttissues. IGF-I stimulates cell differentiation and cell proliferation,and is required by most mammalian cell types for sustainedproliferation. These cell types include, among others, human diploidfibroblasts, epithelial cells, smooth muscle cells, T lymphocytes,neural cells, myeloid cells, chondrocytes, osteoblasts and bone marrowstem cells. For a review of the wide variety of cell types for whichIGF-I/IGF-I receptor interaction mediates cell proliferation, seeGoldring et al., Eukar. Gene Express., 1:31-326 (1991).

The first step in the transduction pathway leading to IGF-I-stimulatedcellular proliferation or differentiation is binding of IGF-I or IGF-II(or insulin at supraphysiological concentrations) to the IGF-I receptor.The IGF-I receptor is composed of two types of subunits: an alphasubunit (a 130-135 kD protein that is entirely extracellular andfunctions in ligand binding) and a beta subunit (a 95-kD transmembraneprotein, with transmembrane and cytoplasmic domains). The IGF-IR belongsto the family of tyrosine kinase growth factor receptors (Ullrich etal., Cell 61: 203-212, 1990), and is structurally similar to the insulinreceptor (Ullrich et al., EMBO J. 5: 2503-2512, 1986). The IGF-IR isinitially synthesized as a single chain proreceptor polypeptide which isprocessed by glycosylation, proteolytic cleavage, and covalent bondingto assemble into a mature 460-kD heterotetramer comprising twoalpha-subunits and two beta-subunits. The beta subunit(s) possessesligand-activated tyrosine kinase activity. This activity is implicatedin the signaling pathways mediating ligand action which involveautophosphorylation of the beta-subunit and phosphorylation of IGF-IRsubstrates.

In vivo, serum levels of IGF-I are dependent upon the presence ofpituitary growth hormone (GH). Although the liver is a major site ofGH-dependent IGF-I synthesis, recent work indicates that the majority ofnormal tissues also produce IGF-I. A variety of neoplastic tissues mayalso produce IGF-I. Thus IGF-I may act as a regulator of normal andabnormal cellular proliferation via autocrine or paracrine, as well asendocrine mechanisms. IGF-I and IGF-II bind to IGF binding proteins(IGFBPs) in vivo. The availability of free IGF for interaction with theIGF-IR is modulated by the IGFBPs. For a review of IGFBPs and IGF-I, seeGrimberg et al., J. Cell. Physiol. 183: 1-9, 2000.

There is considerable evidence for a role for IGF-I and/or IGF-IR in themaintenance of tumor cells in vitro and in vivo. IGF-IR levels areelevated in tumors of lung (Kaiser et al., J. Cancer Res. Clin Oncol.119: 665-668, 1993; Moody et al., Life Sciences 52: 1161-1173, 1993;Macauley et al., Cancer Res., 50: 2511-2517, 1990), breast (Pollak etal., Cancer Lett. 38: 223-230, 1987; Foekens et al., Cancer Res. 49:7002-7009, 1989; Cullen et al., Cancer Res. 49: 7002-7009, 1990; Arteagaet al., J. Clin. Invest. 84: 1418-1423, 1989), prostate and colon(Remaole-Bennet et al., J. Clin. Endocrinol. Metab. 75: 609-616, 1992;Guo et al., Gastroenterol. 102: 1101-1108, 1992). Deregulated expressionof IGF-I in prostate epithelium leads to neoplasia in transgenic mice(DiGiovanni et al., Proc. Natl. Acad. Sci. USA 97: 3455-60, 2000). Inaddition, IGF-I appears to be an autocrine stimulator of human gliomas(Sandberg-Nordqvist et al., Cancer Res. 53: 2475-2478, 1993), whileIGF-I stimulated the growth of fibrosarcomas that overexpressed IGF-IR(Butler et al., Cancer Res. 58: 3021-27, 1998). Further, individualswith “high normal” levels of IGF-I have an increased risk of commoncancers compared to individuals with IGF-I levels in the “low normal”range (Rosen et al., Trends Endocrinol. Metab. 10: 136-41, 1999). Manyof these tumor cell types respond to IGF-I with a proliferative signalin culture (Nakanishi et al., J. Clin. Invest. 82: 354-359, 1988; Freedet al., J. Mol. Endocrinol. 3: 509-514, 1989), and autocrine orparacrine loops for proliferation in vivo have been postulated (LeRoithet al., Endocrine Revs. 16: 143-163, 1995; Yee et al., Mol. Endocrinol.3: 509-514, 1989). For a review of the role IGF-I/IGF-I receptorinteraction plays in the growth of a variety of human tumors, seeMacaulay, Br. J. Cancer, 65: 311-320, 1992.

Increased IGF-I levels are also correlated with several noncancerouspathological states, including acromegaly and gigantism (Barkan,Cleveland Clin. J. Med. 65: 343, 347-349, 1998), while abnormalIGF-I/IGF-I receptor function has been implicated in psoriasis (Wraightet al., Nat. Biotech. 18: 521-526, 2000), atherosclerosis and smoothmuscle restenosis of blood vessels following angioplasty (Bayes-Genis etal., Circ. Res. 86: 125-130, 2000). Increased IGF-I levels also can be aproblem in diabetes or in complications thereof, such as microvascularproliferation (Smith et al., Nat. Med. 5: 1390-1395, 1999). DecreasedIGF-I levels, which occur, inter alia, in cases when GH serum levels aredecreased or when there is an insensitivity or resistance to GH, isassociated with such disorders as small stature (Laron, Paediatr. Drugs1: 155-159, 1999), neuropathy, decrease in muscle mass and osteoporosis(Rosen et al., Trends Endocrinol. Metab. 10: 136-141, 1999).

Using antisense expression vectors or antisense oligonucleotides to theIGF-IR RNA, it has been shown that interference with IGF-IR leads toinhibition of IGF-1-mediated or IGF-II-mediated cell growth (see, e.g.,Wraight et al., Nat. Biotech. 18: 521-526, 2000). The antisense strategywas successful in inhibiting cellular proliferation in several normalcell types and in human tumor cell lines. Growth can also be inhibitedusing peptide analogues of IGF-I (Pietrzkowski et al., Cell Growth &Diff. 3: 199-205, 1992; and Pietrzkowski et al., Mol. Cell. Biol., 12:3883-3889, 1992), or a vector expressing an antisense RNA to the IGF-IRNA (Trojan et al., Science 259: 94-97, 1992). In addition, antibodiesto IGF-IR (Arteaga et al., Breast Canc. Res. Treatm., 22: 101-106, 1992;and Kalebic et al., Cancer Res. 54: 5531-5534, 1994), and dominantnegative mutants of IGF-IR (Prager et al., Proc. Natl. Acad. Sci. U.S.A.91: 2181-2185, 1994; Li et al., J. Biol. Chem., 269: 32558-32564, 1994and Jiang et al., Oncogene 18: 6071-77, 1999), can reverse thetransformed phenotype, inhibit tumorigenesis, and induce loss of themetastatic phenotype.

IGF-I is also important in the regulation of apoptosis. Apoptosis, whichis programmed cell death, is involved in a wide variety of developmentalprocesses, including immune and nervous system maturation. In additionto its role in development, apoptosis also has been implicated as animportant cellular safeguard against tumorigenesis (Williams, Cell 65:1097-1098, 1991; Lane, Nature 362: 786-787, 1993). Suppression of theapoptotic program, by a variety of genetic lesions, may contribute tothe development and progression of malignancies.

IGF-I protects from apoptosis induced by cytokine withdrawal inIL-3-dependent hemopoietic cells (Rodriguez-Tarduchy, G. et al., J.Immunol. 149: 535-540, 1992), and from serum withdrawal in Rat-1/mycERcells (Harrington, E., et al., EMBO J. 13: 3286-3295, 1994). Theanti-apoptotic function of IGF-I is important in the post-commitmentstage of the cell cycle and also in cells blocked in cell cycleprogression by etoposide or thymidine. The demonstration that c-mycdriven fibroblasts are dependent on IGF-I for their survival suggeststhat there is an important role for the IGF-IR in the maintenance oftumor cells by specifically inhibiting apoptosis, a role distinct fromthe proliferative effects of IGF-I or IGF-IR. This would be similar to arole thought to be played by other anti-apoptotic genes such as bcl-2 inpromoting tumor survival (McDonnell et al., Cell 57: 79-88, 1989;Hockenberry et al., Nature 348: 334-336, 1990).

The protective effects of IGF-I on apoptosis are dependent upon havingIGF-IR present on cells to interact with IGF-I (Resnicoff et al., CancerRes. 55: 3739-3741, 1995). Support for an anti-apoptotic function ofIGF-IR in the maintenance of tumor cells was also provided by a studyusing antisense oligonucleotides to the IGF-IR that identified aquantitative relationship between IGF-IR levels, the extent of apoptosisand the tumorigenic potential of a rat syngeneic tumor (Rescinoff etal., Cancer Res. 55: 3739-3741, 1995). An overexpressed IGF-IR has beenfound to protect tumor cells in vitro from etoposide-induced apoptosis(Sell et al., Cancer Res. 55: 303-306, 1995) and, even moredramatically, that a decrease in IGF-IR levels below wild type levelscaused massive apoptosis of tumor cells in vivo (Resnicoff et al.,Cancer Res. 55: 2463-2469, 1995).

Potential strategies for inducing apoptosis or for inhibiting cellproliferation associated with increased IGF-I, increased IGF-II and/orincreased IGF-IR receptor levels include suppressing IGF-I levels orIGF-II levels or preventing the binding of IGF-I to the IGF-IR. Forexample, the long acting somatostatin analogue octreotide has beenemployed to reduce IGF synthesis and/or secretion. Soluble IGF-IR hasbeen used to induce apoptosis in tumor cells in vivo and inhibittumorigenesis in an experimental animal system (D'Ambrosio et al.,Cancer Res. 56: 4013-20, 1996). In addition, IGF-IR antisenseoligonucleotides, peptide analogues of IGF-I, and antibodies to IGF-IRhave been used to decrease IGF-I or IGF-IR expression (see supra).However, none of these compounds has been suitable for long-termadministration to human patients. In addition, although IGF-I has beenadministered to patients for treatment of short stature, osteoporosis,decreased muscle mass, neuropathy or diabetes, the binding of IGF-I toIGFBPs has often made treatment with IGF-I difficult or ineffective.

Accordingly, in view of the roles that IGF-I and IGF-IR have in suchdisorders as cancer and other proliferative disorders when IGF-I and/orIGF-IR are overexpressed, and the roles that too little IGF-I and IGF-IRhave in disorders such as short stature and frailty when either IGF-Iand/or IGF-IR are underexpressed, it would be desirable to generateantibodies to IGF-IR that could be used to either inhibit or stimulateIGF-IR. Although anti-IGF-IR antibodies have been reported to have beenfound in certain patients with autoimmune diseases, none of theseantibodies has been purified and none has been shown to be suitable forinhibiting IGF-I activity for diagnostic or clinical procedures. See,e.g., Thompson et al., Pediat. Res. 32: 455-459, 1988; Tappy et al.,Diabetes 37: 1708-1714, 1988; Weightman et al., Autoimmunity 16:251-257,1993; Drexhage et al., Nether. J. of Med. 45:285-293, 1994. Thus, itwould be desirable to obtain high-affinity human anti-IGF-IR antibodiesthat could be used to treat diseases in humans.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show alignments of the nucleotide sequences of the lightchain variable regions from six human anti-IGF-IR antibodies to eachother and to germline sequences. FIG. 1A shows the alignment of thenucleotide sequences of the variable region of the light chain (VL) ofantibodies 2.12.1 (SEQ ID NO: 1) 2.13.2 (SEQ ID NO: 5), 2.1-4.3 (SEQ IDNO: 9) and 4.9.2 (SEQ ID NO: 13) to each other and to the germline VκA30 sequence (SEQ ID NO: 39). FIG. 1B shows the alignment of thenucleotide sequence of VL of antibody 4.17.3 (SEQ ID NO: 17) to thegermline Vκ 012 sequence (SEQ ID NO: 41). FIG. 1C shows the alignment ofthe nucleotide sequence of VL of antibody 6.1.1 (SEQ ID NO: 21) to thegermline Vκ A27 sequence (SEQ ID NO: 37). The alignments also show theCDR regions of the VL from each antibody. The consensus sequences forFIGS. 1A-1C are shown in SEQ ID NOS: 53-55, respectively.

FIGS. 2A-2D show alignments of the nucleotide sequences of the heavychain variable regions from six human anti-IGF-IR antibodies to eachother and to germline sequences. FIG. 2A shows the alignment of thenucleotide sequence of the VH of antibody 2.12.1 (SEQ ID NO: 3) to thegermline VH DP-35 sequence (SEQ ID NO: 29). FIG. 2B shows the alignmentof the nucleotide sequence of the VH of antibody 2.14.3 (SEQ ID NO: 11)to the germline VIV-4/4.35 sequence (SEQ ID NO: 43). FIGS. 2C-1 and 2C-2show the alignments of the nucleotide sequences of the VH of antibodies2.13.2 (SEQ ID NO: 7), 4.9.2 (SEQ ID NO: 15) and 6.1.1 (SEQ ID NO: 23)to each other and to the germline VH DP-47 sequence (SEQ ID NO: 31).FIG. 2D shows the alignment of the nucleotide sequence of the VH ofantibody 4.17.3 (SEQ ID NO: 19) to the germline VH DP-71 sequence (SEQID NO: 35). The alignment also shows the CDR regions of the antibodies.The consensus sequences for FIGS. 2A-2D are shown in SEQ ID NOS: 56-59,respectively.

FIG. 3 shows that anti-IGF-IR antibodies 2.13.2, 4.9.2 and 2.12.1inhibit IGF-I binding to 3T3-IGF-IR cells.

FIG. 4 shows that anti-IGF-IR antibody 4.9.2 inhibits IGF-I-inducedreceptor tyrosine phosphorylation (upper panel) and induces IGF-IRdownregulation at the cell surface (lower panel).

FIG. 5 shows that anti-IGF-IR antibodies 2.13.2 and 4.9.2 reduce IGF-IRphosphotyrosine signal in 3T3-IGF-IR tumors.

FIG. 6 shows that anti-IGF-IR antibodies 2.13.2 and 4.9.2 reduce IGF-IRin 3T3-IGF-IR tumors.

FIG. 7 shows that anti-IGF-IR antibody 2.13.2 inhibits 3T3-IGF-IR tumorgrowth in vivo alone (left panel) or in combination with adriamycin(right panel).

FIG. 8 shows the relationship between anti-IGF-IR antibody 2.13.2 serumlevels and IGF-IR downregulation in 3T3-IGF-IR tumors.

FIG. 9 shows that multiple doses of anti-IGF-IR antibody 2.13.2 inhibit3T3-IGF-IR tumor growth in vivo alone or in combination with adriamycin.

FIG. 10 shows that anti-IGF-IR antibody 2.13.2 inhibits large tumorgrowth in vivo in combination with adriamycin.

FIG. 11 shows that anti-IGF-IR antibody 2.13.2 inhibits Colo 205 tumorgrowth in vivo alone or in combination with 5-deoxyuridine (5-FU).

FIG. 12 shows that multiple doses of anti-IGF-IR antibody 2.13.2 inhibitColo 205 tumor growth in vivo alone or in combination with 5-FU.

FIG. 13 shows that multiple doses of anti-IGF-IR antibody 2.13.2 inhibitMCF-7 tumor growth in vivo alone or in combination with taxol.

FIG. 14 shows that anti-IGF-IR antibody 2.13.2 inhibits MCF-7 tumorgrowth in vivo alone (left panel) or in combination with adriamycin(right panel).

FIG. 15 shows that multiple doses of anti-IGF-IR antibody 2.13.2 inhibitMCF-7 tumor growth in vivo alone or in combination with tamoxifen.

FIG. 16 shows that multiple doses of anti-IGF-IR antibody 2.13.2 inhibitA431 tumor growth in vivo alone or in combination with the epidermalgrowth factor receptor (EGF-R) tyrosine kinase inhibitor CP-358,774.

FIG. 17 shows a pharmacokinetic evaluation of a single intravenousinjection of anti-IGF-IR antibody 2.13.2 in Cynomologus monkeys.

FIG. 18 shows that the combination of anti-IGF-IR antibody 2.13.2 andadriamycin increases the downregulation of IGF-IR on 3T3-IGF-IR tumorsin vivo.

FIG. 19A shows the number of mutations in different regions of the heavyand light chains of 2.13.2 and 2.12.1 compared to the germlinesequences.

FIGS. 19B-E show alignments of the amino acid sequences from the heavyand light chains of antibodies 2.13.2 and 2.12.1 with the germlinesequences from which they are derived. FIG. 19B shows an alignment ofthe amino acid sequence of the heavy chain of antibody 2.13.2 (SEQ IDNO: 45) with that of germline sequence DP-47(3-23)/D6-19/JH6 (SEQ ID NO:46). FIG. 19C shows an alignment of the amino acid sequence of the lightchain of antibody 2.13.2 (SEQ ID NO: 47) with that of germline sequenceA30/Jk2 (SEQ ID NO: 48). FIG. 19D shows an alignment of the amino acidsequence of the heavy chain of antibody 2.12.1 (SEQ ID NO: 49) with thatof germline sequence DP-35(3-11)/D3-3/JH6 (SEQ ID NO: 50). FIG. 19Eshows an alignment of the amino acid sequence of the light chain ofantibody 2.12.1 (SEQ ID NO: 51) with that of germline sequence A30/Jk1(SEQ ID NO: 52). For FIGS. 19B-E, the signal sequences are in italic,the CDRs are underlined, the variable domains are bold, the framework(FR) mutations are highlighted with a plus sign (“+”) above the aminoacid residue and CDR mutations are highlighted with an asterisk abovethe amino acid residue.

SUMMARY OF THE INVENTION

The present invention provides an isolated antibody or antigen-bindingportion thereof that binds IGF-IR, preferably one that binds to primateand human IGF-IR, and more preferably one that is a human antibody. Theinvention provides an anti-IGF-IR antibody that inhibits the binding ofIGF-I or IGF-II to IGF-IR, and also provides an anti-IGF-IR antibodythat activates IGF-IR.

The invention provides a pharmaceutical composition comprising theantibody and a pharmaceutically acceptable carrier. The pharmaceuticalcomposition may further comprise another component, such as ananti-tumor agent or an imaging reagent.

Diagnostic and therapeutic methods are also provided by the invention.Diagnostic methods include a method for diagnosing the presence orlocation of an IGF-IR-expressing tissue using an anti-IGF-IR antibody. Atherapeutic method comprises administering the antibody to a subject inneed thereof, preferably in conjunction with administration of anothertherapeutic agent.

The invention provides an isolated cell line, such as a hybridoma, thatproduces an anti-IGF-IR antibody.

The invention also provides nucleic acid molecules encoding the heavyand/or light chain or antigen-binding portions thereof of an anti-IGF-IRantibody.

The invention provides vectors and host cells comprising the nucleicacid molecules, as well as methods of recombinantly producing thepolypeptides encoded by the nucleic acid molecules.

Non-human transgenic animals that express the heavy and/or light chainor antigen-binding portions thereof of an anti-IGF-IR antibody are alsoprovided. The invention also provides a method for treating a subject inneed thereof with an effective amount of a nucleic acid moleculeencoding the heavy and/or light chain or antigen-binding portionsthereof of an anti-IGF-IR antibody.

DETAILED DESCRIPTION OF THE INVENTION Definitions and General Techniques

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those well known and commonly used in the art. The methodsand techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al. Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates (1992), and Harlow and Lane Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1990), which are incorporated herein by reference.Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications, as commonly accomplished in the art oras described herein. The nomenclatures used in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well known and commonly used in the art. Standardtechniques are used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofpatients.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

The term “polypeptide” encompasses native or artificial proteins,protein fragments and polypeptide analogs of a protein sequence. Apolypeptide may be monomeric or polymeric.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation (1) isnot associated with naturally associated components that accompany it inits native state, (2) is free of other proteins from the same species(3) is expressed by a cell from a different species, or (4) does notoccur in nature. Thus, a polypeptide that is chemically synthesized orsynthesized in a cellular system different from the cell from which itnaturally originates will be “isolated” from its naturally associatedcomponents. A protein may also be rendered substantially free ofnaturally associated components by isolation, using protein purificationtechniques well known in the art.

A protein or polypeptide is “substantially pure,” “substantiallyhomogeneous” or “substantially purified” when at least about 60 to 75%of a sample exhibits a single species of polypeptide. The polypeptide orprotein may be monomeric or multimeric. A substantially pure polypeptideor protein will typically comprise about 50%, 60, 70%, 80% or 90% W/W ofa protein sample, more usually about 95%, and preferably will be over99% pure. Protein purity or homogeneity may be indicated by a number ofmeans well known in the art, such as polyacrylamide gel electrophoresisof a protein sample, followed by visualizing a single polypeptide bandupon staining the gel with a stain well known in the art. For certainpurposes, higher resolution may be provided by using HPLC or other meanswell known in the art for purification.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence. Fragments typically areat least 5, 6, 8 or 10 amino acids long, preferably at least 14 aminoacids long, more preferably at least 20 amino acids long, usually atleast 50 amino acids long, even more preferably at least 70, 80, 90,100, 150 or 200 amino acids long.

The term “polypeptide analog” as used herein refers to a polypeptidethat is comprised of a segment of at least 25 amino acids that hassubstantial identity to a portion of an amino acid sequence and that hasat least one of the following properties: (1) specific binding to IGF-IRunder suitable binding conditions, (2) ability to block IGF-I or IGF-IIbinding to IGF-IR, or (3) ability to reduce IGF-IR cell surfaceexpression or tyrosine phosphorylation in vitro or in vivo. Typically,polypeptide analogs comprise a conservative amino acid substitution (orinsertion or deletion) with respect to the naturally-occurring sequence.Analogs typically are at least 20 amino acids long, preferably at least50, 60, 70, 80, 90, 100, 150 or 200 amino acids long or longer, and canoften be as long as a full-length naturally-occurring polypeptide.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W.H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et al. Nature 354:105 (1991), which are each incorporatedherein by reference.

Non-peptide analogs are commonly used in the pharmaceutical industry asdrugs with properties analogous to those of the template peptide. Thesetypes of non-peptide compound are termed “peptide mimetics” or“peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber andFreidinger TINS p. 392 (1985); and Evans et al. J. Med. Chem. 30:1229(1987), which are incorporated herein by reference. Such compounds areoften developed with the aid of computerized molecular modeling. Peptidemimetics that are structurally similar to therapeutically usefulpeptides may be used to produce an equivalent therapeutic orprophylactic effect. Generally, peptidomimetics are structurally similarto a paradigm polypeptide (i.e., a polypeptide that has a desiredbiochemical property or pharmacological activity), such as a humanantibody, but have one or more peptide linkages optionally replaced by alinkage selected from the group consisting of: —CH₂NH—, —CH₂S—,—CH₂—CH₂—, —CH═CH-(cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, bymethods well known in the art. Systematic substitution of one or moreamino acids of a consensus sequence with a D-amino acid of the same type(e.g., D-lysine in place of L-lysine) may also be used to generate morestable peptides. In addition, constrained peptides comprising aconsensus sequence or a substantially identical consensus sequencevariation may be generated by methods known in the art (Rizo andGierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference); for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.

An “immunoglobulin” is a tetrameric molecule. In a naturally-occurringimmunoglobulin, each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The amino-terminal portion of eachchain includes a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The carboxy-terminalportion of each chain defines a constant region primarily responsiblefor effector function. Human light chains are classified as κ and λlight chains. Heavy chains are classified as μ, Δ, γ, α, or ε, anddefine the antibody's isotype as IgM, IgD, IgG, IgA, and IgE,respectively. Within light and heavy chains, the variable and constantregions are joined by a “J” region of about 12 or more amino acids, withthe heavy chain also including a “D” region of about 10 more aminoacids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nded. Raven Press, N.Y. (1989)) (incorporated by reference in its entiretyfor all purposes). The variable regions of each light/heavy chain pairform the antibody binding site such that an intact immunoglobulin hastwo binding sites.

Immunoglobulin chains exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hypervariable regions,also called complementarity determining regions or CDRs. The CDRs fromthe two chains of each pair are aligned by the framework regions,enabling binding to a specific epitope. From N-terminus to C-terminus,both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each domain is inaccordance with the definitions of Kabat Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.(1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987);Chothia et al. Nature 342:878-883 (1989).

An “antibody” refers to an intact immunoglobulin or to anantigen-binding portion thereof that competes with the intact antibodyfor specific binding. Antigen-binding portions may be produced byrecombinant DNA techniques or by enzymatic or chemical cleavage ofintact antibodies. Antigen-binding portions include, inter alia, Fab,Fab′, F(ab′)₂, Fv, dAb, and complementarity determining region (CDR)fragments, single-chain antibodies (scFv), chimeric antibodies,diabodies and polypeptides that contain at least a portion of animmunoglobulin that is sufficient to confer specific antigen binding tothe polypeptide.

As used herein, an antibody that is referred to as, e.g., 2.12.1,2.13.2, 2.14.3, 4.9.2, 4.17.3 and 6.1.1, is an antibody that is derivedfrom the hybridoma of the same name. For example, antibody 2.12.1 isderived from hybridoma 2.12.1.

An Fab fragment is a monovalent fragment consisting of the VL, VH, CLand CH I domains; a F(ab′)₂ fragment is a bivalent fragment comprisingtwo Fab fragments linked by a disulfide bridge at the hinge region; a Fdfragment consists of the VH and CH1 domains; an Fv fragment consists ofthe VL and VH domains of a single arm of an antibody; and a dAb fragment(Ward et al., Nature 341:544-546, 1989) consists of a VH domain.

A single-chain antibody (scFv) is an antibody in which a VL and VHregions are paired to form a monovalent molecules via a synthetic linkerthat enables them to be made as a single protein chain (Bird et al.,Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA85:5879-5883, 1988). Diabodies are bivalent, bispecific antibodies inwhich VH and VL domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see e.g., Holliger, P., et al., Proc. Natl. Acad. Sci. USA90:6444-6448, 1993, and Poljak, R. J., et al., Structure 2:1121-1123,1994). One or more CDRs may be incorporated into a molecule eithercovalently or noncovalently to make it an immunoadhesin. Animmunoadhesin may incorporate the CDR(s) as part of a larger polypeptidechain, may covalently link the CDR(s) to another polypeptide chain, ormay incorporate the CDR(s) noncovalently. The CDRs permit theimmunoadhesin to specifically bind to a particular antigen of interest.

An antibody may have one or more binding sites. If there is more thanone binding site, the binding sites may be identical to one another ormay be different. For instance, a naturally-occurring immunoglobulin hastwo identical binding sites, a single-chain antibody or Fab fragment hasone binding site, while a “bispecific” or “bifunctional” antibody hastwo different binding sites.

An “isolated antibody” is an antibody that (1) is not associated withnaturally-associated components, including other naturally-associatedantibodies, that accompany it in its native state, (2) is free of otherproteins from the same species, (3) is expressed by a cell from adifferent species, or (4) does not occur in nature. Examples of isolatedantibodies include an anti-IGF-IR antibody that has been affinitypurified using IGF-IR is an isolated antibody, an anti-IGF-IR antibodythat has been synthesized by a hybridoma or other cell line in vitro,and a human anti-IGF-IR antibody derived from a transgenic mouse.

The term “human antibody” includes all antibodies that have one or morevariable and constant regions derived from human immunoglobulinsequences. In a preferred embodiment, all of the variable and constantdomains are derived from human immunoglobulin sequences (a fully humanantibody). These antibodies may be prepared in a variety of ways, asdescribed below.

A humanized antibody is an antibody that is derived from a non-humanspecies, in which certain amino acids in the framework and constantdomains of the heavy and light chains have been mutated so as to avoidor abrogate an immune response in humans. Alternatively, a humanizedantibody may be produced by fusing the constant domains from a humanantibody to the variable domains of a non-human species. Examples of howto make humanized antibodies may be found in U.S. Pat. Nos. 6,054,297,5,886,152 and 5,877,293.

The term “chimeric antibody” refers to an antibody that contains one ormore regions from one antibody and one or more regions from one or moreother antibodies. In a preferred embodiment, one or more of the CDRs arederived from a human anti-IGF-IR antibody. In a more preferredembodiment, all of the CDRs are derived from a human anti-IGF-IRantibody. In another preferred embodiment, the CDRs from more than onehuman anti-IGF-IR antibodies are mixed and matched in a chimericantibody. For instance, a chimeric antibody may comprise a CDR1 from thelight chain of a first human anti-IGF-IR antibody may be combined withCDR2 and CDR3 from the light chain of a second human anti-IGF-IRantibody, and the CDRs from the heavy chain may be derived from a thirdanti-IGF-IR antibody. Further, the framework regions may be derived fromone of the same anti-IGF-IR antibodies, from one or more differentantibodies, such as a human antibody, or from a humanized antibody.

A “neutralizing antibody” or “an inhibitory antibody” is an antibodythat inhibits the binding of IGF-IR to IGF-I when an excess of theanti-IGF-IR antibody reduces the amount of IGF-I bound to IGF-IR by atleast about 20%. In a preferred embodiment, the antibody reduces theamount of IGF-I bound to IGF-IR by at least 40%, more preferably 60%,even more preferably 80%, or even more preferably 85%. The bindingreduction may be measured by any means known to one of ordinary skill inthe art, for example, as measured in an in vitro competitive bindingassay. An example of measuring the reduction in binding of IGF-I toIGF-IR is presented below in Example IV.

An “activating antibody” is an antibody that activates IGF-IR by atleast about 20% when added to a cell, tissue or organism expressingIGF-IR. In a preferred embodiment, the antibody activates IGF-IRactivity by at least 40%, more preferably 60%, even more preferably 80%,or even more preferably 85%. In a more preferred embodiment, theactivating antibody is added in the presence of IGF-I or IGF-II. Inanother preferred embodiment, the activity of the activating antibody ismeasured by determining the amount of tyrosine autophosphorylation ofIGF-IR.

Fragments or analogs of antibodies can be readily prepared by those ofordinary skill in the art following the teachings of this specification.Preferred amino- and carboxy-termini of fragments or analogs occur nearboundaries of functional domains. Structural and functional domains canbe identified by comparison of the nucleotide and/or amino acid sequencedata to public or proprietary sequence databases. Preferably,computerized comparison methods are used to identify sequence motifs orpredicted protein conformation domains that occur in other proteins ofknown structure and/or function. Methods to identify protein sequencesthat fold into a known three-dimensional structure are known. Bowie etal. Science 253:164 (1991).

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jonsson, U., et al. (1993) Ann. Biol. Clin.51:19-26; Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson,B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al.(1991) Anal. Biochem. 198:268-277.

The term “K_(off)” refers to the off rate constant for dissociation ofan antibody from the antibody/antigen complex.

The term “K_(d)” refers to the dissociation constant of a particularantibody-antigen interaction.

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor. Epitopic determinantsusually consist of chemically active surface groupings of molecules suchas amino acids or sugar side chains and usually have specific threedimensional structural characteristics, as well as specific chargecharacteristics. An antibody is said to specifically bind an antigenwhen the dissociation constant is ≦1 μM, preferably ≦100 nM and mostpreferably ≦10 nM.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), which is incorporated herein by reference.Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α-, α-disubstituted amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidsmay also be suitable components for polypeptides of the presentinvention. Examples of unconventional amino acids include:4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, s-N-methylarginine, and othersimilar amino acids and imino acids (e.g., 4-hydroxyproline). In thepolypeptide notation used herein, the lefthand direction is the aminoterminal direction and the righthand direction is the carboxy-terminaldirection, in accordance with standard usage and convention.

The term “polynucleotide” as referred to herein means a polymeric formof nucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms of DNA.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence.

The term “oligonucleotide” referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. Preferably oligonucleotides are 10 to 60bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or20 to 40 bases in length. Oligonucleotides are usually single stranded,e.g. for probes; although oligonucleotides may be double stranded, e.g.for use in the construction of a gene mutant. Oligonucleotides of theinvention can be either sense or antisense oligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoroamidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984);Stein et al. Nucl. Acids Res. 16:3209 (1988); Zon et al. Anti-CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990), the disclosures ofwhich are hereby incorporated by reference. An oligonucleotide caninclude a label for detection, if desired.

“Operably linked” sequences include both expression control sequencesthat are contiguous with the gene of interest and expression controlsequences that act in trans or at a distance to control the gene ofinterest. The term “expression control sequence” as used herein refersto polynucleotide sequences which are necessary to effect the expressionand processing of coding sequences to which they are ligated. Expressioncontrol sequences include appropriate transcription initiation,termination, promoter and enhancer sequences; efficient RNA processingsignals such as splicing and polyadenylation signals; sequences thatstabilize cytoplasmic mRNA; sequences that enhance translationefficiency (i.e., Kozak consensus sequence); sequences that enhanceprotein stability; and when desired, sequences that enhance proteinsecretion. The nature of such control sequences differs depending uponthe host organism; in prokaryotes, such control sequences generallyinclude promoter, ribosomal binding site, and transcription terminationsequence; in eukaryotes, generally, such control sequences includepromoters and transcription termination sequence. The term “controlsequences” is intended to include, at a minimum, all components whosepresence is essential for expression and processing, and can alsoinclude additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

The term “selectively hybridize” referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsthereof in accordance with the invention selectively hybridize tonucleic acid strands under hybridization and wash conditions thatminimize appreciable amounts of detectable binding to nonspecificnucleic acids. “High stringency” or “highly stringent” conditions can beused to achieve selective hybridization conditions as known in the artand discussed herein. An example of “high stringency” or “highlystringent” conditions is a method of incubating a polynucleotide withanother polynucleotide, wherein one polynucleotide may be affixed to asolid surface such as a membrane, in a hybridization buffer of 6×SSPE orSSC, 50% formamide, 5×Denhardt's reagent, 0.5% SDS, 100 μg/ml denatured,fragmented salmon sperm DNA at a hybridization temperature of 42° C. for12-16 hours, followed by twice washing at 55° C. using a wash buffer of1×SSC, 0.5% SDS. See also Sambrook et al., supra, pp. 9.50-9.55.

The term “percent sequence identity” in the context of nucleic acidsequences refers to the residues in two sequences which are the samewhen aligned for maximum correspondence. The length of sequence identitycomparison may be over a stretch of at least about nine nucleotides,usually at least about 18 nucleotides, more usually at least about 24nucleotides, typically at least about 28 nucleotides, more typically atleast about 32 nucleotides, and preferably at least about 36, 48 or morenucleotides. There are a number of different algorithms known in the artwhich can be used to measure nucleotide sequence identity. For instance,polynucleotide sequences can be compared using FASTA, Gap or Bestfit,which are programs in Wisconsin Package Version 10.0, Genetics ComputerGroup (GCG), Madison, Wis. FASTA, which includes, e.g., the programsFASTA2 and FASTA3, provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson, Methods Enzymol. 183: 63-98 (1990); Pearson, Methods Mol.Biol. 132: 185-219 (2000); Pearson, Methods Enzymol. 266: 227-258(1996); Pearson, J. Mol. Biol. 276: 71-84 (1998); herein incorporated byreference). Unless otherwise specified, default parameters for aparticular program or algorithm are used. For instance, percent sequenceidentity between nucleic acid sequences can be determined using FASTAwith its default parameters (a word size of 6 and the NOPAM factor forthe scoring matrix) or using Gap with its default parameters as providedin GCG Version 6.1, herein incorporated by reference.

A reference to a nucleic acid sequence encompasses its complement unlessotherwise specified. Thus, a reference to a nucleic acid molecule havinga particular sequence should be understood to encompass itscomplementary strand, with its complementary sequence.

In the molecular biology art, researchers use the terms “percentsequence identity”, “percent sequence similarity” and “percent sequencehomology” interchangeably. In this application, these terms shall havethe same meaning with respect to nucleic acid sequences only.

The term “substantial similarity” or “substantial sequence similarity,”when referring to a nucleic acid or fragment thereof, indicates that,when optimally aligned with appropriate nucleotide insertions ordeletions with another nucleic acid (or its complementary strand), thereis nucleotide sequence identity in at least about 85%, preferably atleast about 90%, and more preferably at least about 95%, 96%, 97%, 98%or 99% of the nucleotide bases, as measured by any well-known algorithmof sequence identity, such as FASTA, BLAST or Gap, as discussed above.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 75% or 80%sequence identity, preferably at least 90% or 95% sequence identity,even more preferably at least 98% or 99% sequence identity. Preferably,residue positions which are not identical differ by conservative aminoacid substitutions. A “conservative amino acid substitution” is one inwhich an amino acid residue is substituted by another amino acid residuehaving a side chain (R group) with similar chemical properties (e.g.,charge or hydrophobicity). In general, a conservative amino acidsubstitution will not substantially change the functional properties ofa protein. In cases where two or more amino acid sequences differ fromeach other by conservative substitutions, the percent sequence identityor degree of similarity may be adjusted upwards to correct for theconservative nature of the substitution. Means for making thisadjustment are well-known to those of skill in the art. See, e.g.,Pearson, Methods Mol. Biol. 24: 307-31 (1994), herein incorporated byreference. Examples of groups of amino acids that have side chains withsimilar chemical properties include 1) aliphatic side chains: glycine,alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl sidechains: serine and threonine; 3) amide-containing side chains:asparagine and glutamine; 4) aromatic side chains: phenylalanine,tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, andhistidine; and 6) sulfur-containing side chains are cysteine andmethionine. Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.

Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al., Science 256: 1443-45 (1992), herein incorporated by reference. A“moderately conservative” replacement is any change having a nonnegativevalue in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG contains programs such as “Gap” and “Bestfit” whichcan be used with default parameters to determine sequence homology orsequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson (1990); Pearson (2000). Another preferred algorithm whencomparing a sequence of the invention to a database containing a largenumber of sequences from different organisms is the computer programBLAST, especially blastp or tblastn, using default parameters. See,e.g., Altschul et al., J. Mol. Biol. 215: 403-410 (1990); Altschul etal., Nucleic Acids Res. 25:3389-402 (1997); herein incorporated byreference.

The length of polypeptide sequences compared for homology will generallybe at least about 16 amino acid residues, usually at least about 20residues, more usually at least about 24 residues, typically at leastabout 28 residues, and preferably more than about 35 residues. Whensearching a database containing sequences from a large number ofdifferent organisms, it is preferable to compare amino acid sequences.

As used herein, the terms “label” or “labeled” refers to incorporationof another molecule in the antibody. In one embodiment, the label is adetectable marker, e.g., incorporation of a radiolabeled amino acid orattachment to a polypeptide of biotinyl moieties that can be detected bymarked avidin (e.g., streptavidin containing a fluorescent marker orenzymatic activity that can be detected by optical or colorimetricmethods). In another embodiment, the label or marker can be therapeutic,e.g., a drug conjugate or toxin. Various methods of labelingpolypeptides and glycoproteins are known in the art and may be used.Examples of labels for polypeptides include, but are not limited to, thefollowing: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescentmarkers, biotinyl groups, predetermined polypeptide epitopes recognizedby a secondary reporter (e.g., leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags),magnetic agents, such as gadolinium chelates, toxins such as pertussistoxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. In some embodiments, labels are attached by spacerarms of various lengths to reduce potential steric hindrance.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials. The term “pharmaceutical agent or drug” asused herein refers to a chemical compound or composition capable ofinducing a desired therapeutic effect when properly administered to apatient. Other chemistry terms herein are used according to conventionalusage in the art, as exemplified by The McGraw-Hill Dictionary ofChemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)),incorporated herein by reference).

The term “antineoplastic agent” is used herein to refer to agents thathave the functional property of inhibiting a development or progressionof a neoplasm in a human, particularly a malignant (cancerous) lesion,such as a carcinoma, sarcoma, lymphoma, or leukemia Inhibition ofmetastasis is frequently a property of antineoplastic agents.

The term patient includes human and veterinary subjects.

Human Anti-IGF-IR Antibodies and Characterization Thereof

Human antibodies avoid certain of the problems associated withantibodies that possess mouse or rat variable and/or constant regions.The presence of such mouse or rat derived sequences can lead to therapid clearance of the antibodies or can lead to the generation of animmune response against the antibody by a patient. Therefore, in oneembodiment, the invention provides humanized anti-IGF-IR antibodies. Ina preferred embodiment, the invention provides fully human anti-IGF-IRantibodies by introducing human immunoglobulin genes into a rodent sothat the rodent produces fully human antibodies. More preferred arefully human anti-human IGF-IR antibodies. Fully human anti-IGF-IRantibodies are expected to minimize the immunogenic and allergicresponses intrinsic to mouse or mouse-derivatized monoclonal antibodies(Mabs) and thus to increase the efficacy and safety of the administeredantibodies. The use of fully human antibodies can be expected to providea substantial advantage in the treatment of chronic and recurring humandiseases, such as inflammation and cancer, which may require repeatedantibody administrations. In another embodiment, the invention providesan anti-IGF-IR antibody that does not bind complement.

In a preferred embodiment, the anti-IGF-IR antibody is 2.12.1, 2.13.2,2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. In another preferred embodiment,the anti-IGF-IR antibody comprises a light chain comprising an aminoacid sequence selected from SEQ ID NO: 2, 6, 10, 14, 18 or 22, or one ormore CDRs from these amino acid sequences. In another preferredembodiment, the anti-IGF-IR antibody comprises a heavy chain comprisingan amino acid sequence selected from SEQ ID NO: 4, 8, 12, 16, 20 or 24or one or more CDRs from these amino acid sequences.

Class and Subclass of Anti-IGF-IR Antibodies

The antibody may be an IgG, an IgM, an IgE, an IgA or an IgD molecule.In a preferred embodiment, the antibody is an IgG and is an IgG1, IgG2,IgG3 or IgG4 subtype. In a more preferred embodiment, the anti-IGF-IRantibody is subclass IgG2. In another preferred embodiment, theanti-IGF-IR antibody is the same class and subclass as antibody 2.12.1,2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1, which is IgG2.

The class and subclass of anti-IGF-IR antibodies may be determined byany method known in the art. In general, the class and subclass of anantibody may be determined using antibodies that are specific for aparticular class and subclass of antibody. Such antibodies are availablecommercially. The class and subclass can be determined by ELISA, WesternBlot as well as other techniques. Alternatively, the class and subclassmay be determined by sequencing all or a portion of the constant domainsof the heavy and/or light chains of the antibodies, comparing theiramino acid sequences to the known amino acid sequences of various classand subclasses of immunoglobulins, and determining the class andsubclass of the antibodies.

Species and Molecule Selectivity

In another aspect of the invention, the anti-IGF-IR antibodydemonstrates both species and molecule selectivity. In one embodiment,the anti-IGF-IR antibody binds to human, cynomologous or rhesus IGF-IR.In a preferred embodiment, the anti-IGF-IR antibody does not bind tomouse, rat, guinea pig, dog or rabbit IGF-IR. In another preferredembodiment, the anti-IGF-IR antibody does not bind to a New World monkeyspecies such as a marmoset. Following the teachings of thespecification, one may determine the species selectivity for theanti-IGF-IR antibody using methods well known in the art. For instance,one may determine species selectivity using Western blot, FACS, ELISA orRIA. In a preferred embodiment, one may determine the speciesselectivity using Western blot.

In another embodiment, the anti-IGF-IR antibody has a selectivity forIGF-IR that is at least 50 times greater than its selectivity forinsulin receptor. In a preferred embodiment, the selectivity of theanti-IGF-IR antibody is more than 100 times greater than its selectivityfor insulin receptor. In an even more preferred embodiment, theanti-IGF-IR antibody does not exhibit any appreciable specific bindingto any other protein other than IGF-IR. One may determine theselectivity of the anti-IGF-IR antibody for IGR-IR using methods wellknown in the art following the teachings of the specification. Forinstance, one may determine the selectivity using Western blot, FACS,ELISA or RIA. In a preferred embodiment, one may determine the molecularselectivity using Western blot.

Binding Affinity of Anti-IGF-IR to IGF-IR

In another aspect of the invention, the anti-IGF-IR antibodies bind toIGF-IR with high affinity. In one embodiment, the anti-IGF-IR antibodybinds to IGF-IR with a K_(d) of 1×10⁻⁸ M or less. In a more preferredembodiment, the antibody binds to IGF-IR with a K_(a) or 1×10⁻⁹ M orless. In an even more preferred embodiment, the antibody binds to IGF-IRwith a K_(d) or 5×10⁻¹⁰ M or less. In another preferred embodiment, theantibody binds to IGF-IR with a K_(d) or 1×10⁻¹⁰ M or less. In anotherpreferred embodiment, the antibody binds to IGF-IR with substantiallythe same K_(d) as an antibody selected from 2.12.1, 2.13.2, 2.14.3,3.1.1, 4.9.2, 4.17.3 or 6.1.1. In another preferred embodiment, theantibody binds to IGF-IR with substantially the same K_(d) as anantibody that comprises one or more CDRs from an antibody selected from2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. In still anotherpreferred embodiment, the antibody binds to IGF-IR with substantiallythe same K_(d) as an antibody that comprises one of the amino acidsequences selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22 or 24. In another preferred embodiment, the antibody binds to IGF-IRwith substantially the same K_(d) as an antibody that comprises one ormore CDRs from an antibody that comprises one of the amino acidsequences selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22 or 24.

In another aspect of the invention, the anti-IGF-IR antibody has a lowdissociation rate. In one embodiment, the anti-IGF-IR antibody has anK_(off) of 1×10⁻⁴ s⁻¹ or lower. In a preferred embodiment, the K_(off)is 5×10⁻⁵ s⁻¹ or lower. In another preferred embodiment, the K_(off) issubstantially the same as an antibody selected from 2.12.1, 2.13.2,2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. In another preferred embodiment,the antibody binds to IGF-IR with substantially the same K_(off) as anantibody that comprises one or more CDRs from an antibody selected from2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. In still anotherpreferred embodiment, the antibody binds to IGF-IR with substantiallythe same K_(off) as an antibody that comprises one of the amino acidsequences selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22 or 24. In another preferred embodiment, the antibody binds to IGF-IRwith substantially the same K_(off) as an antibody that comprises one ormore CDRs from an antibody that comprises one of the amino acidsequences selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22.

The binding affinity and dissociation rate of an anti-IGF-IR antibody toIGF-IR may be determined by any method known in the art. In oneembodiment, the binding affinity can be measured by competitive ELISAs,RIAs or surface plasmon resonance, such as BIAcore. The dissociationrate can also be measured by surface plasmon resonance. In a morepreferred embodiment, the binding affinity and dissociation rate ismeasured by surface plasmon resonance. In an even more preferredembodiment, the binding affinity and dissociation rate is measured usinga BIAcore. An example of determining binding affinity and dissociationrate is described below in Example II.

Half-Life of Anti-IGF-IR Antibodies

According to another object of the invention, the anti-IGF-IR antibodyhas a half-life of at least one day in vitro or in vivo. In a preferredembodiment, the antibody or portion thereof has a half-life of at leastthree days. In a more preferred embodiment, the antibody or portionthereof has a half-life of four days or longer. In another embodiment,the antibody or portion thereof has a half-life of eight days or longer.In another embodiment, the antibody or antigen-binding portion thereofis derivatized or modified such that it has a longer half-life, asdiscussed below. In another preferred embodiment, the antibody maycontain point mutations to increase serum half life, such as describedWO 00/09560, published Feb. 24, 2000.

The antibody half-life may be measured by any means known to one havingordinary skill in the art. For instance, the antibody half life may bemeasured by Western blot, ELISA or RIA over an appropriate period oftime. The antibody half-life may be measured in any appropriate animals,e.g., a monkey, such as a cynomologous monkey, a primate or a human.

Identification of IGF-IR Epitopes Recognized by Anti-IGF-IR Antibody

The invention also provides an anti-IGF-IR antibody that binds the sameantigen or epitope as a human anti-IGF-IR antibody. Further, theinvention provides an anti-IGF-IR antibody that cross-competes with ahuman anti-IGF-IR antibody. In a preferred embodiment, the humananti-IGF-IR antibody is 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or6.1.1. In another preferred embodiment, the human anti-IGF-IR comprisesone or more CDRs from an antibody selected from 2.12.1, 2.13.2, 2.14.3,3.1.1, 4.9.2, 4.17.3 or 6.1.1. In still another preferred embodiment,the human anti-IGF-IR comprises one of the amino acid sequences selectedfrom SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24. Inanother preferred embodiment, the human anti-IGF-IR comprises one ormore CDRs from an antibody that comprises one of the amino acidsequences selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22 or 24. In a highly preferred embodiment, the anti-IGF-IR antibody isanother human antibody.

One may determine whether an anti-IGF-IR antibody binds to the sameantigen using a variety of methods known in the art. For instance, onemay determine whether a test anti-IGF-IR antibody binds to the sameantigen by using an anti-IGF-IR antibody to capture an antigen that isknown to bind to the anti-IGF-IR antibody, such as IGF-IR, eluting theantigen from the antibody, and then determining whether the testantibody will bind to the eluted antigen. One may determine whether anantibody binds to the same epitope as an anti-IGF-IR antibody by bindingthe anti-IGF-IR antibody to IGF-IR under saturating conditions, and thenmeasuring the ability of the test antibody to bind to IGF-IR. If thetest antibody is able to bind to the IGF-IR at the same time as theanti-IGF-IR antibody, then the test antibody binds to a differentepitope as the anti-IGF-IR antibody. However, if the test antibody isnot able to bind to the IGF-IR at the same time, then the test antibodybinds to the same epitope as the human anti-IGF-IR antibody. Thisexperiment may be performed using ELISA, RIA or surface plasmonresonance. In a preferred embodiment, the experiment is performed usingsurface plasmon resonance. In a more preferred embodiment, BIAcore isused. One may also determine whether an anti-IGF-IR antibodycross-competes with an anti-IGF-IR antibody. In a preferred embodiment,one may determine whether an anti-IGF-IR antibody cross-competes withanother by using the same method that is used to measure whether theanti-IGF-IR antibody is able to bind to the same epitope as anotheranti-IGF-IR antibody.

Light and Heavy Chain Usage

The invention also provides an anti-IGF-IR antibody that comprisesvariable sequences encoded by a human κ gene. In a preferred embodiment,the variable sequences are encoded by either the Vκ A27, A30 or O12 genefamily. In a preferred embodiment, the variable sequences are encoded bya human Vκ A30 gene family. In a more preferred embodiment, the lightchain comprises no more than ten amino acid substitutions from thegermline Vκ A27, A30 or O12, preferably no more than six amino acidsubstitutions, and more preferably no more than three amino acidsubstitutions. In a preferred embodiment, the amino acid substitutionsare conservative substitutions.

SEQ ID NOS: 2, 6, 10, 14, 18 and 22 provide the amino acid sequences ofthe variable regions of six anti-IGF-IR κ light chains. SEQ ID NOS: 38,40 and 42 provide the amino acid sequences of the three germline κ lightchains from which the six anti-IGF-IR κ light chains are derived. FIGS.1A-1C show the alignments of the nucleotide sequences of the variableregions of the light chains of the six anti-IGF-IR antibodies to eachother and to the germline sequences from which they are derived.Following the teachings of this specification, one of ordinary skill inthe art could determine the encoded amino acid sequence of the sixanti-IGF-IR κ light chains and the germline κ light chains and determinethe differences between the germline sequences and the antibodysequences.

In a preferred embodiment, the VL of the anti-IGF-IR antibody containsthe same amino acid substitutions, relative to the germline amino acidsequence, as any one or more of the VL of antibodies 2.12.1, 2.13.2,2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. For example, the VL of theanti-IGF-IR antibody may contain one or more amino acid substitutionsthat are the same as those present in antibody 2.13.2, another aminoacid substitution that is the same as that present in antibody 2.14.3,and another amino acid substitution that is the same as antibody 4.9.2.In this manner, one can mix and match different features of antibodybinding in order to alter, e.g., the affinity of the antibody for IGF-IRor its dissociation rate from the antigen. In another embodiment, theamino acid substitutions are made in the same position as those found inany one or more of the VL of antibodies 2.12.1, 2.13.2, 2.14.3, 3.1.1,4.9.2, 4.17.3 or 6.1.1, but conservative amino acid substitutions aremade rather than using the same amino acid. For example, if the aminoacid substitution compared to the germline in one of the antibodies2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1 is glutamate, onemay conservatively substitute aspartate. Similarly, if the amino acidsubstitution is serine, one may conservatively substitute threonine.

In another preferred embodiment, the light chain comprises an amino acidsequence that is the same as the amino acid sequence of the VL of2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. In another highlypreferred embodiment, the light chain comprises amino acid sequencesthat are the same as the CDR regions of the light chain of 2.12.1,2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. In another preferredembodiment, the light chain comprises an amino acid sequence from atleast one CDR region of the light chain of 2.12.1, 2.13.2, 2.14.3,3.1.1, 4.9.2, 4.17.3 or 6.1.1. In another preferred embodiment, thelight chain comprises amino acid sequences from CDRs from differentlight chains. In a more preferred embodiment, the CDRs from differentlight chains are obtained from 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2,4.17.3 or 6.1.1. In another preferred embodiment, the light chaincomprises an amino acid sequence selected from SEQ ID NOS: 2, 6, 10, 14,18 or 22. In another embodiment, the light chain comprises an amino acidsequence encoded by a nucleic acid sequence selected from SEQ ID NOS: 1,5, 9, 13, 17 or 21, or a nucleic acid sequence that encodes an aminoacid sequence having 1-10 amino acid insertions, deletions orsubstitutions therefrom. Preferably, the amino acid substitutions areconservative amino acid substitutions. In another embodiment, theantibody or portion thereof comprises a lambda light chain.

The present invention also provides an anti-IGF-IR antibody or portionthereof comprises a human heavy chain or a sequence derived from a humanheavy chain. In one embodiment, the heavy chain amino acid sequence isderived from a human V_(H) DP-35, DP-47, DP-70, DP-71 or VIV-4/4.35 genefamily. In a preferred embodiment, the heavy chain amino acid sequenceis derived from a human V_(H) DP-47 gene family. In a more preferredembodiment, the heavy chain comprises no more than eight amino acidchanges from germline V_(H) DP-35, DP-47, DP-70, DP-71 or VIV-4/4.35,more preferably no more than six amino acid changes, and even morepreferably no more than three amino acid changes.

SEQ ID NOS: 4, 8, 12, 16, 20 and 24 provide the amino acid sequences ofthe variable regions of six anti-IGF-IR heavy chains. SEQ ID NOS: 30,32, 34, 36 and 44 provide the amino acid sequences and SEQ ID NOS: 29,31, 33, 35 and 43 provide the nucleotide sequences of the germline heavychains DP-35, DP-47, DP-70, DP-71 and VIV-4, respectively. FIGS. 2A-2Dshow the alignments of the amino acid sequences of the variable regionof the six anti-IGF-IR antibodies to their corresponding germlinesequences. Following the teachings of this specification, one ofordinary skill in the art could determine the encoded amino acidsequence of the six anti-IGF-IR heavy chains and the germline heavychains and determine the differences between the germline sequences andthe antibody sequences.

In a preferred embodiment, the VH of the anti-IGF-IR antibody containsthe same amino acid substitutions, relative to the germline amino acidsequence, as any one or more of the VH of antibodies 2.12.1, 2.13.2,2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. Similar to what was discussedabove, the VH of the anti-IGF-IR antibody may contain one or more aminoacid substitutions that are the same as those present in antibody2.13.2, another amino acid substitution that is the same as that presentin antibody 2.14.3, and another amino acid substitution that is the sameas antibody 4.9.2. In this manner, one can mix and match differentfeatures of antibody binding in order to alter, e.g., the affinity ofthe antibody for IGF-IR or its dissociation rate from the antigen. Inanother embodiment, the amino acid substitutions are made in the sameposition as those found in any one or more of the VH of antibodies2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.17.3., 4.9.2 or 6.1.1, but conservativeamino acid substitutions are made rather than using the same amino acid.

In another preferred embodiment, the heavy chain comprises an amino acidsequence that is the same as the amino acid sequence of the VH of2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. In another highlypreferred embodiment, the heavy chain comprises amino acid sequencesthat are the same as the CDR regions of the heavy chain of 2.12.1,2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. In another preferredembodiment, the heavy chain comprises an amino acid sequence from atleast one CDR region of the heavy chain of 2.12.1, 2.13.2, 2.14.3,3.1.1, 4.9.2, 4.17.3 or 6.1.1. In another preferred embodiment, theheavy chain comprises amino acid sequences from CDRs from differentheavy chains. In a more preferred embodiment, the CDRs from differentheavy chains are obtained from 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2,4.17.3 or 6.1.1. In another preferred embodiment, the heavy chaincomprises an amino acid sequence selected from SEQ ID NOS: 4, 8, 12, 16,20 or 24. In another embodiment, the heavy chain comprises an amino acidsequence encoded by a nucleic acid sequence selected from SEQ ID NOS: 3,7, 11, 15, 19 or 23, or a nucleic acid sequence that encodes an aminoacid sequence having 1-10 amino acid insertions, deletions orsubstitutions therefrom. In another embodiment, the substitutions areconservative amino acid substitutions.

Inhibition of IGF-IR Activity by Anti-IGF-IR Antibody Inhibition ofIGF-I Binding to IGF-IR

In another embodiment, the invention provides an anti-IGF-IR antibodythat inhibits the binding of IGF-I to IGF-IR or the binding of IGF-II toIGF-IR. In a preferred embodiment, the IGF-IR is human. In anotherpreferred embodiment, the anti-IGF-IR antibody is a human antibody. Inanother embodiment, the antibody or portion thereof inhibits bindingbetween IGF-IR and IGF-I with an IC₅₀ of no more than 100 nM. In apreferred embodiment, the IC₅₀ is no more than 10 nM. In a morepreferred embodiment, the IC₅₀ is no more than 5 nM. The IC₅₀ can bemeasured by any method known in the art. Typically, an IC₅₀ can bemeasured by ELISA or RIA. In a preferred embodiment, the IC₅₀ ismeasured by RIA.

In another embodiment, the invention provides an anti-IGF-IR antibodythat prevents activation of the IGF-IR in the presence of IGF-I. In apreferred embodiment, the anti-IGF-IR antibody inhibits IGF-IR-inducedtyrosine phosphorylation that occurs upon occupancy of the receptor. Inanother preferred embodiment, the anti-IGF-IR antibody inhibitsdownstream cellular events from occurring. For instance, the anti-IGF-IRcan inhibit tyrosine phosphorylation of Shc and insulin receptorsubstrate (IRS) 1 and 2, all of which are normally phosphorylated whencells are treated with IGF-I (Kim et al., J. Biol. Chem. 273:34543-34550, 1998). One can determine whether an anti-IGF-IR antibodycan prevent activation of IGF-IR in the presence of IGF-1 by determiningthe levels of autophosphorylation for IGF-IR, Shc, IRS-1 or IRS-2 byWestern blot or immunopreciptation. In a preferred embodiment, one woulddetermine the levels of autophosphorylation of IGF-IR by Western blot.See, e.g., Example VII.

In another aspect of the invention, the antibody causes thedownregulation of IGF-IR from a cell treated with the antibody. In oneembodiment, the IGF-IR is internalized into the cytoplasm of the cell.After the anti-IGF-IR antibody binds to IGF-IR, the antibody isinternalized, as shown by confocal microscopy. Without wishing to bebound to any theory, it is believed that the antibody-IGF-IR complex isinternalized into a lysosome and degraded. One may measure thedownregulation of IGF-IR by any method known in the art includingimmunoprecipitation, confocal microscopy or Western blot. See, e.g.,Example VII. In a preferred embodiment, the antibody is selected 2.12.1,2.13.2, 2.14.3, 3.1.1, 4.9.2, or 6.1.1, or comprises a heavy chain,light chain or antigen-binding region thereof.

Activation of IGF-IR by Anti-IGF-IR Antibody

Another aspect of the present invention involves activating anti-IGF-IRantibodies. An activating antibody differs from an inhibiting antibodybecause it amplifies or substitutes for the effects of IGF-I on IGF-IR.In one embodiment, the activating antibody is able to bind to IGF-IR andcause it to be activated in the absence of IGF-I. This type ofactivating antibody is essentially a mimic of IGF-I. In anotherembodiment, the activating antibody amplifies the effect of IGF-I onIGF-IR. This type of antibody does not activate IGF-IR by itself, butrather increases the activation of IGF-IR in the presence of IGF-I. Amimic anti-IGF-IR antibody may be easily distinguished from anamplifying anti-IGF-IR antibody by treating cells in vitro with anantibody in the presence or absence of low levels of IGF-I. If theantibody is able to cause IGF-IR activation in the absence of IGF-I,e.g., it increases IGF-IR tyrosine phosphorylation, then the antibody isa mimic antibody. If the antibody cannot cause IGF-IR activation in theabsence of IGF-I but is able to amplify the amount of IGF-IR activation,then the antibody is an amplifying antibody. In a preferred embodiment,the activating antibody is 4.17.3. In another preferred embodiment, theantibody comprises one or more CDRs from 4.17.3. In another preferredembodiment, the antibody is derived from either or both of the germlinesequences 012 (light chain) and/or D71 (heavy chain).

Inhibition of IGF-IR Tyrosine Phosphorylation, IGF-IR Levels and TumorCell Growth In Vivo by Anti-IGF-IR Antibodies

Another embodiment of the invention provides an anti-IGF-IR antibodythat inhibits IGF-IR tyrosine phosphorylation and receptor levels invivo. In one embodiment, administration of anti-IGF-IR antibody to ananimal causes a reduction in IGF-IR phosphotyrosine signal inIGF-IR-expressing tumors. In a preferred embodiment, the anti-IGF-IRantibody causes a reduction in phosphotyrosine signal by at least 20%.In a more preferred embodiment, the anti-IGF-IR antibody causes adecrease in phosphotyrosine signal by at least 60%, more preferably 50%.In an even more preferred embodiment, the antibody causes a decrease inphosphotyrosine signal of at least 40%, more preferably 30%, even morepreferably 20%. In a preferred embodiment, the antibody is administeredapproximately 24 hours before the levels of tyrosine phosphorylation aremeasured. The levels of tyrosine phosphorylation may be measured by anymethod known in the art, such as those described infra. See, e.g.,Example III and FIG. 5. In a preferred embodiment, the antibody isselected from 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, or 6.1.1, orcomprises a heavy chain, light chain or antigen-binding portion thereof.

In another embodiment, administration of anti-IGF-IR antibody to ananimal causes a reduction in IGF-IR levels in IGF-IR-expressing tumors.In a preferred embodiment, the anti-IGF-IR antibody causes a reductionin receptor levels by at least 20% compared to an untreated animal. In amore preferred embodiment, the anti-IGF-IR antibody causes a decrease inreceptor levels to at least 60%, more preferably 50% of the receptorlevels in an untreated animal. In an even more preferred embodiment, theantibody causes a decrease in receptor levels to at least 40%, morepreferably 30%. In a preferred embodiment, the antibody is administeredapproximately 24 hours before the IGF-IR levels are measured. The IGF-IRlevels may be measured by any method known in the art, such as thosedescribed infra. See, e.g., Example VIII and FIG. 6. In a preferredembodiment, the antibody is selected from 2.12.1, 2.13.2, 2.14.3, 3.1.1,4.9.2, or 6.1.1, or comprises a heavy chain, light chain orantigen-binding portion thereof.

In another embodiment, an anti-IGF-IR antibody inhibits tumor cellgrowth in vivo. The tumor cell may be derived from any cell typeincluding, without limitation, epidermal, epithelial, endothelial,leukemia, sarcoma, multiple myeloma or mesodermal cells. Examples oftumor cells include A549 (non-small cell lung carcinoma) cells, MCF-7cells, Colo 205 cells, 3T3/IGF-IR cells and A431 cells. In a preferredembodiment, the antibody inhibits tumor cell growth as compared to thegrowth of the tumor in an untreated animal. In a more preferredembodiment, the antibody inhibits tumor cell growth by 50%. In an evenmore preferred embodiment, the antibody inhibits tumor cell growth by60%, 65%, 70% or 75%. In one embodiment, the inhibition of tumor cellgrowth is measured at least 7 days after the animals have startedtreatment with the antibody. In a more preferred embodiment, theinhibition of tumor cell growth is measured at least 14 days after theanimals have started treatment with the antibody. In another preferredembodiment, another antineoplastic agent is administered to the animalwith the anti-IGF-IR antibody. In a preferred embodiment, theantineoplastic agent is able to further inhibit tumor cell growth. In aneven more preferred embodiment, the antineoplastic agent is adriamycin,taxol, tamoxifen, 5-fluorodeoxyuridine (5-FU) or CP-358,774. In apreferred embodiment, the co-administration of an antineoplastic agentand the anti-IGF-IR antibody inhibits tumor cell growth by at least 50%,more preferably 60%, 65%, 70% or 75%, more preferably 80%, 85% or 90%after a period of 22-24 days. See, e.g., FIG. 7 and Example IX. In apreferred embodiment, the antibody is selected from 2.12.1, 2.13.2,2.14.3, 3.1.1, 4.9.2, or 6.1.1, or comprises a heavy chain, light chainor antigen-binding portion thereof.

Induction of Apoptosis by Anti-IGF-IR Antibodies

Another aspect of the invention provides an anti-IGF-IR antibody thatinduces cell death. In one embodiment, the antibody causes apoptosis.The antibody may induce apoptosis either in vivo or in vitro. Ingeneral, tumor cells are more sensitive to apoptosis than normal cells,such that administration of an anti-IGF-IR antibody causes apoptosis ofa tumor cell preferentially to that of a normal cell. In anotherembodiment, the administration of an anti-IGF-IR antibody decreaseslevels of an enzyme, akt, which is involved in the phosphatidyl inositol(PI) kinase pathway. The PI kinase pathway, in turn, is involved in thecell proliferation and prevention of apoptosis. Thus, inhibition of aktcan cause apoptosis. In a more preferred embodiment, the antibody isadministered in vivo to cause apoptosis of an IGF-IR-expressing cell. Ina preferred embodiment, the antibody is selected from 2.12.1, 2.13.2,2.14.3, 3.1.1, 4.9.2, or 6.1.1, or comprises a heavy chain, light chainor antigen-binding portion thereof.

Methods of Producing Antibodies and Antibody-Producing Cell LinesImmunization

In one embodiment of the instant invention, human antibodies areproduced by immunizing a non-human animal comprising some or all of thehuman immunoglobulin locus with an IGF-IR antigen. In a preferredembodiment, the non-human animal is a XENOMOUSE™, which is an engineeredmouse strain that comprises large fragments of the human immunoglobulinloci and is deficient in mouse antibody production. See, e.g., Green etal. Nature Genetics 7:13-21 (1994) and U.S. Pat. Nos. 5,916,771,5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598 and6,130,364. See also WO 91/10741, published Jul. 25, 1991, WO 94/02602,published Feb. 3, 1994, WO 96/34096 and WO 96/33735, both published Oct.31, 1996, WO 98/16654, published Apr. 23, 1998, WO 98/24893, publishedJun. 11, 1998, WO 98/50433, published Nov. 12, 1998, WO 99/45031,published Sep. 10, 1999, WO 99/53049, published Oct. 21, 1999, WO 0009560, published Feb. 24, 2000 and WO 00/037504, published Jun. 29,2000. The XENOMOUSE™ produces an adult-like human repertoire of fullyhuman antibodies, and generates antigen-specific human Mabs. A secondgeneration XENOMOUSE™ contains approximately 80% of the human antibodyrepertoire through introduction of megabase sized, germlineconfiguration YAC fragments of the human heavy chain loci and κ lightchain loci. See Mendez et al. Nature Genetics 15:146-156 (1997), Greenand Jakobovits J. Exp. Med. 188:483-495 (1998), the disclosures of whichare hereby incorporated by reference.

The invention also provides a method for making anti-IGF-IR antibodiesfrom non-human, non-mouse animals by immunizing non-human transgenicanimals that comprise human immunoglobulin loci. One may produce suchanimals using the methods described immediately above. The methodsdisclosed in these patents may modified as described in U.S. Pat. No.5,994,619. In a preferred embodiment, the non-human animals may be rats,sheep, pigs, goats, cattle or horses.

In another embodiment, the non-human animal comprising humanimmunoglobulin gene loci are animals that have a “minilocus” of humanimmunoglobulins. In the minilocus approach, an exogenous Ig locus ismimicked through the inclusion of individual genes from the Ig locus.Thus, one or more V_(H) genes, one or more D_(H) genes, one or moreJ_(H) genes, a mu constant region, and a second constant region(preferably a gamma constant region) are formed into a construct forinsertion into an animal. This approach is described, inter alia, inU.S. Pat. Nos. 5,545,807, 5,545,806, 5,625,825, 5,625,126, 5,633,425,5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,591,669, 5,612,205,5,721,367, 5,789,215, and 5,643,763, hereby incorporated by reference.

An advantage of the minilocus approach is the rapidity with whichconstructs including portions of the Ig locus can be generated andintroduced into animals. However, a potential disadvantage of theminilocus approach is that there may not be sufficient immunoglobulindiversity to support full B-cell development, such that there may belower antibody production.

In order to produce a human anti-IGF-IR antibody, a non-human animalcomprising some or all of the human immunoglobulin loci is immunizedwith an IGF-IR antigen and the antibody or the antibody-producing cellis isolated from the animal. The IGF-IR antigen may be isolated and/orpurified IGF-IR and is preferably a human IGF-IR. In another embodiment,the IGF-IR antigen is a fragment of IGF-IR, preferably the extracellulardomain of IGF-IR. In another embodiment, the IGF-IR antigen is afragment that comprises at least one epitope of IGF-IR. In anotherembodiment, the IGF-IR antigen is a cell that expresses IGF-IR on itscell surface, preferably a cell that overexpresses IGF-IR on its cellsurface.

Immunization of animals may be done by any method known in the art. See,e.g., Harlow and Lane, Antibodies: A Laboratory Manual, New York: ColdSpring Harbor Press, 1990. Methods for immunizing non-human animals suchas mice, rats, sheep, goats, pigs, cattle and horses are well known inthe art. See, e.g., Harlow and Lane and U.S. Pat. No. 5,994,619. In apreferred embodiment, the IGF-IR antigen is administered with a adjuvantto stimulate the immune response. Such adjuvants include complete orincomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM(immunostimulating complexes). Such adjuvants may protect thepolypeptide from rapid dispersal by sequestering it in a local deposit,or they may contain substances that stimulate the host to secretefactors that are chemotactic for macrophages and other components of theimmune system. Preferably, if a polypeptide is being administered, theimmunization schedule will involve two or more administrations of thepolypeptide, spread out over several weeks.

Example I provides an protocol for immunizing a XENOMOUSE™ withfull-length human IGF-IR in phosphate-buffered saline.

Production of Antibodies and Antibody-Producing Cell Lines

After immunization of an animal with an IGF-IR antigen, antibodiesand/or antibody-producing cells may be obtained from the animal. Ananti-IGF-IR antibody-containing serum is obtained from the animal bybleeding or sacrificing the animal. The serum may be used as it isobtained from the animal, an immunoglobulin fraction may be obtainedfrom the serum, or the anti-IGF-IR antibodies may be purified from theserum. Serum or immunoglobulins obtained in this manner are polyclonal,which are disadvantageous because the amount of antibodies that can beobtained is limited and the polyclonal antibody has a heterogeneousarray of properties.

In another embodiment, antibody-producing immortalized hybridomas may beprepared from the immunized animal. After immunization, the animal issacrificed and the splenic B cells are fused to immortalized myelomacells as is well-known in the art. See, e.g., Harlow and Lane, supra. Ina preferred embodiment, the myeloma cells do not secrete immunoglobulinpolypeptides (a non-secretory cell line). After fusion and antibioticselection, the hybridomas are screened using IGF-IR, a portion thereof,or a cell expressing IGF-IR. In a preferred embodiment, the initialscreening is performed using an enzyme-linked immunoassay (ELISA) or aradioimmunoassay (RIA), preferably an ELISA. An example of ELISAscreening is provided in WO 00/37504, herein incorporated by reference.

In another embodiment, antibody-producing cells may be prepared from ahuman who has an autoimmune disorder and who expresses anti-IGF-IRantibodies. Cells expressing the anti-IGF-IR antibodies may be isolatedby isolating white blood cells and subjecting them tofluorescence-activated cell sorting (FACS) or by panning on platescoated with IGF-IR or a portion thereof. These cells may be fused with ahuman non-secretory myeloma to produce human hybridomas expressing humananti-IGF-IR antibodies. In general, this is a less preferred embodimentbecause it is likely that the anti-IGF-IR antibodies will have a lowaffinity for IGF-IR.

Anti-IGF-IR antibody-producing hybridomas are selected, cloned andfurther screened for desirable characteristics, including robusthybridoma growth, high antibody production and desirable antibodycharacteristics, as discussed further below. Hybridomas may be culturedand expanded in vivo in syngeneic animals, in animals that lack animmune system, e.g., nude mice, or in cell culture in vitro. Methods ofselecting, cloning and expanding hybridomas are well known to those ofordinary skill in the art.

Preferably, the immunized animal is a non-human animal that expresseshuman immunoglobulin genes and the splenic B cells are fused to amyeloma derived from the same species as the non-human animal. Morepreferably, the immunized animal is a XENOMOUSE™ and the myeloma cellline is a non-secretory mouse myeloma, such as the myeloma cell line isNSO-bcl2. See, e.g., Example I.

In one aspect, the invention provides hybridomas are produced thatproduce human anti-IGF-IR antibodies. In a preferred embodiment, thehybridomas are mouse hybridomas, as described above. In anotherpreferred embodiment, the hybridomas are produced in a non-human,non-mouse species such as rats, sheep, pigs, goats, cattle or horses. Inanother embodiment, the hybridomas are human hybridomas, in which ahuman non-secretory myeloma is fused with a human cell expressing ananti-IGF-IR antibody.

Nucleic Acids, Vectors, Host Cells and Recombinant Methods of MakingAntibodies Nucleic Acids

Nucleic acid molecules encoding anti-IGF-IR antibodies of the inventionare provided. In one embodiment, the nucleic acid molecule encodes aheavy and/or light chain of an anti-IGF-IR immunoglobulin. In apreferred embodiment, a single nucleic acid molecule encodes a heavychain of an anti-IGF-IR immunoglobulin and another nucleic acid moleculeencodes the light chain of an anti-IGF-IR immunoglobulin. In a morepreferred embodiment, the encoded immunoglobulin is a humanimmunoglobulin, preferably a human IgG. The encoded light chain may be aλ chain or a κ chain, preferably a κ chain.

The nucleic acid molecule encoding the variable region of the lightchain may be derived from the A30, A27 or O12 Vκ gene. In a preferredembodiment, the light chain is derived from the A30 Vκ gene. In anotherpreferred embodiment, the nucleic acid molecule encoding the light chaincomprises the joining region derived from Jκ1, Jκ2 or Jκ4. In an evenmore preferred embodiment, the nucleic acid molecule encoding the lightchain contains no more than ten amino acid changes from the germline A30Vκ gene, preferably no more than six amino acid changes, and even morepreferably no more than three amino acid changes.

The invention provides a nucleic acid molecule that encodes a variableregion of the light chain (VL) containing at least three amino acidchanges compared to the germline sequence, wherein the amino acidchanges are identical to the amino acid changes from the germlinesequence from the VL of one of the antibodies 2.12.1, 2.13.2, 2.14.3,3.1.1, 4.9.2, 4.17.3 or 6.1.1. The invention also provides a nucleicacid molecule comprising a nucleic acid sequence that encodes the aminoacid sequence of the variable region of the light chain of 2.12.1,2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. The invention alsoprovides a nucleic acid molecule comprising a nucleic acid sequence thatencodes the amino acid sequence of one or more of the CDRs of any one ofthe light chains of 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or6.1.1. In a preferred embodiment, the nucleic acid molecule comprises anucleic acid sequence that encodes the amino acid sequence of all of theCDRs of any one of the light chains of 2.12.1, 2.13.2, 2.14.3, 3.1.1,4.9.2, 4.17.3 or 6.1.1. In another embodiment, the nucleic acid moleculecomprises a nucleic acid sequence that encodes the amino acid sequenceof one of SEQ ID NOS: 2, 6, 10, 14, 18 or 22 or comprises a nucleic acidsequence of one of SEQ ID NOS: 1, 5, 9, 13, 17 or 21. In anotherpreferred embodiment, the nucleic acid molecule comprises a nucleic acidsequence that encodes the amino acid sequence of one or more of the CDRsof any one of SEQ ID NOS: 2, 6, 10, 14, 18 or 22 or comprises a nucleicacid sequence of one or more of the CDRs of any one of SEQ ID NOS: 1, 5,9, 13, 17 or 21. In a more preferred embodiment, the nucleic acidmolecule comprises a nucleic acid sequence that encodes the amino acidsequence of all of the CDRs of any one of SEQ ID NOS: 2, 6, 10, 14, 18or 22 or comprises a nucleic acid sequence of all the CDRs of any one ofSEQ ID NOS: 1, 5, 9, 13, 17 or 21.

The invention also provides a nucleic acid molecules that encodes anamino acid sequence of a VL that has an amino acid sequence that is atleast 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to aVL described above, particularly to a VL that comprises an amino acidsequence of one of SEQ ID NOS: 2, 6, 10, 14, 18 or 22. The inventionalso provides a nucleic acid sequence that is at least 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequenceof one of SEQ ID NOS: 1, 5, 9, 13, 17 or 21. In another embodiment, theinvention provides a nucleic acid molecule encoding a VL that hybridizesunder highly stringent conditions to a nucleic acid molecule encoding aVL as described above, particularly a nucleic acid molecule thatcomprises a nucleic acid sequence encoding an amino acid sequence of SEQID NOS: 2, 6, 10, 14, 18 or 22. The invention also provides a nucleicacid sequence encoding an VL that hybridizes under highly stringentconditions to a nucleic acid molecule comprising a nucleic acid sequenceof one of SEQ ID NOS: 1, 5, 9, 13, 17 or 21.

The invention also provides a nucleic acid molecule encoding thevariable region of the heavy chain (VH) is derived from the DP-35,DP-47, DP-71 or VIV-4/4.35 VH gene, preferably the DP-35 VH gene. Inanother preferred embodiment, the nucleic acid molecule encoding the VHcomprises the joining region derived from JH6 or JH5, more preferablyJH6. In another preferred embodiment, the D segment is derived from 3-3,6-19 or 4-17. In an even more preferred embodiment, the nucleic acidmolecule encoding the VH contains no more than ten amino acid changesfrom the germline DP-47 gene, preferably no more than six amino acidchanges, and even more preferably no more than three amino acid changes.In a highly preferred embodiment, the nucleic acid molecule encoding theVH contains at least one amino acid change compared to the germlinesequence, wherein the amino acid change is identical to the amino acidchange from the germline sequence from the heavy chain of one of theantibodies 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. In aneven more preferred embodiment, the VH contains at least three aminoacid changes compared to the germline sequences, wherein the changes areidentical to those changes from the germline sequence from the VH of oneof the antibodies 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1.

In one embodiment, the nucleic acid molecule comprises a nucleic acidsequence that encodes the amino acid sequence of the VH of 2.12.1,2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. In another embodiment,the nucleic acid molecule comprises a nucleic acid sequence that encodesthe amino acid sequence of one or more of the CDRs of the heavy chain of2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. In a preferredembodiment, the nucleic acid molecule comprises a nucleic acid sequencethat encodes the amino acid sequences of all of the CDRs of the heavychain of 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. Inanother preferred embodiment, the nucleic acid molecule comprises anucleic acid sequence that encodes the amino acid sequence of one of SEQID NOS: 4, 8, 12, 16, 20 or 24 or that comprises a nucleic acid sequenceof one of SEQ ID NOS: 3, 7, 11, 15, 19 or 23. In another preferredembodiment, the nucleic acid molecule comprises a nucleic acid sequencethat encodes the amino acid sequence of one or more of the CDRs of anyone of SEQ ID NOS: 4, 8, 12, 16, 20 or 24 or comprises a nucleic acidsequence of one or more of the CDRs of any one of SEQ ID NOS: 3, 7, 11,15, 19 or 23. In a preferred embodiment, the nucleic acid moleculecomprises a nucleic acid sequence that encodes the amino acid sequencesof all of the CDRs of any one of SEQ ID NOS: 4, 8, 12, 16, 20 or 24 orcomprises a nucleic acid sequence of all of the CDRs of any one of SEQID NOS: 3, 7, 11, 15, 19 or 23.

In another embodiment, the nucleic acid molecule encodes an amino acidsequence of a VH that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% identical to one of the amino acid sequences encoding aVH as described immediately above, particularly to a VH that comprisesan amino acid sequence of one of SEQ ID NOS: 4, 8, 12, 16, 20 or 24. Theinvention also provides a nucleic acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleicacid sequence of one of SEQ ID NOS: 3, 7, 11, 15, 19 or 23. In anotherembodiment, the nucleic acid molecule encoding a VH is one thathybridizes under highly stringent conditions to a nucleic acid sequenceencoding a VH as described above, particularly to a VH that comprises anamino acid sequence of one of SEQ ID NOS: 4, 8, 12, 16, 20 or 24. Theinvention also provides a nucleic acid sequence encoding a VH thathybridizes under highly stringent conditions to a nucleic acid moleculecomprising a nucleic acid sequence of one of SEQ ID NOS: 3, 7, 11, 15,19 or 23.

The nucleic acid molecule encoding either or both of the entire heavyand light chains of an anti-IGF-IR antibody or the variable regionsthereof may be obtained from any source that produces an anti-IGF-IRantibody. Methods of isolating mRNA encoding an antibody are well-knownin the art. See, e.g., Sambrook et al. The mRNA may be used to producecDNA for use in the polymerase chain reaction (PCR) or cDNA cloning ofantibody genes. In one embodiment of the invention, the nucleic acidmolecules may be obtained from a hybridoma that expresses an anti-IGF-IRantibody, as described above, preferably a hybridoma that has as one ofits fusion partners a transgenic animal cell that expresses humanimmunoglobulin genes, such as a XENOMOUSE™, non-human mouse transgenicanimal or a non-human, non-mouse transgenic animal. In anotherembodiment, the hybridoma is derived from a non-human, non-transgenicanimal, which may be used, e.g., for humanized antibodies.

A nucleic acid molecule encoding the entire heavy chain of ananti-IGF-IR antibody may be constructed by fusing a nucleic acidmolecule encoding the variable domain of a heavy chain or anantigen-binding domain thereof with a constant domain of a heavy chain.Similarly, a nucleic acid molecule encoding the light chain of ananti-IGF-IR antibody may be constructed by fusing a nucleic acidmolecule encoding the variable domain of a light chain or anantigen-binding domain thereof with a constant domain of a light chain.The nucleic acid molecules encoding the VH and VL chain may be convertedto full-length antibody genes by inserting them into expression vectorsalready encoding heavy chain constant and light chain constant regions,respectively, such that the VH segment is operatively linked to theheavy chain constant region (CH) segment(s) within the vector and the VLsegment is operatively linked to the light chain constant region (CL)segment within the vector. Alternatively, the nucleic acid moleculesencoding the VH or VL chains are converted into full-length antibodygenes by linking, e.g., ligating, the nucleic acid molecule encoding aVH chain to a nucleic acid molecule encoding a CH chain using standardmolecular biological techniques. The same may be achieved using nucleicacid molecules encoding VL and CL chains. The sequences of human heavyand light chain constant region genes are known in the art. See, e.g.,Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.,NIH Publ. No. 91-3242, 1991. Nucleic acid molecules encoding thefull-length heavy and/or light chains may then be expressed from a cellinto which they have been introduced and the anti-IGF-IR antibodyisolated.

In a preferred embodiment, the nucleic acid encoding the variable regionof the heavy chain encodes the amino acid sequence of SEQ ID NOS: 4, 8,12, 16, 20 or 24, and the nucleic acid molecule encoding the variableregion of the light chains encodes the amino acid sequence of SEQ IDNOS: 2, 6, 10, 14, 18 or 22. SEQ ID NO: 28 depicts the amino acidsequence and SEQ ID NO: 27 depicts the nucleic acid sequence encodingthe constant region of the heavy chain of the anti-IGF-IR antibodies2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 and 6.1.1. SEQ ID NO: 26depicts the amino acid sequence and SEQ ID NO: 25 depicts the nucleicacid sequence encoding the constant region of the light chain of theanti-IGF-IR antibodies 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 and6.1.1. Thus, in a preferred embodiment, the nucleic acid moleculeencoding the constant domain of the heavy chain encodes SEQ ID NO: 28,and the nucleic acid molecule encoding the constant domain of the lightchain encodes SEQ ID NO: 26. In a more preferred embodiment, the nucleicacid molecule encoding the constant domain of the heavy chain has thenucleic acid sequence of SEQ ID NO: 27, and the nucleic acid moleculeencoding the constant domain has the nucleic acid sequence of SEQ ID NO:25.

In another embodiment, a nucleic acid molecule encoding either the heavychain of an anti-IGF-IR antibody or an antigen-binding domain thereof,or the light chain of an anti-IGF-IR antibody or an antigen-bindingdomain thereof may be isolated from a non-human, non-mouse animal thatexpresses human immunoglobulin genes and has been immunized with anIGF-IR antigen. In other embodiment, the nucleic acid molecule may beisolated from an anti-IGF-IR antibody-producing cell derived from anon-transgenic animal or from a human patient who produces anti-IGF-IRantibodies. Methods of isolating mRNA from the anti-IGF-IRantibody-producing cells may be isolated by standard techniques, clonedand/or amplified using PCR and library construction techniques, andscreened using standard protocols to obtain nucleic acid moleculesencoding anti-IGF-IR heavy and light chains.

The nucleic acid molecules may be used to recombinantly express largequantities of anti-IGF-IR antibodies, as described below. The nucleicacid molecules may also be used to produce chimeric antibodies, singlechain antibodies, immunoadhesins, diabodies, mutated antibodies andantibody derivatives, as described further below. If the nucleic acidmolecules are derived from a non-human, non-transgenic animal, thenucleic acid molecules may be used for antibody humanization, also asdescribed below.

In another embodiment, the nucleic acid molecules of the invention maybe used as probes or PCR primers for specific antibody sequences. Forinstance, a nucleic acid molecule probe may be used in diagnosticmethods or a nucleic acid molecule PCR primer may be used to amplifyregions of DNA that could be used, inter alia, to isolate nucleic acidsequences for use in producing variable domains of anti-IGF-IRantibodies. In a preferred embodiment, the nucleic acid molecules areoligonucleotides. In a more preferred embodiment, the oligonucleotidesare from highly variable regions of the heavy and light chains of theantibody of interest. In an even more preferred embodiment, theoligonucleotides encode all or a part of one or more of the CDRs.

Vectors

The invention provides vectors comprising the nucleic acid molecules ofthe invention that encode the heavy chain or the antigen-binding portionthereof. The invention also provides vectors comprising the nucleic acidmolecules of the invention that encode the light chain orantigen-binding portion thereof. The invention also provides vectorscomprising nucleic acid molecules encoding fusion proteins, modifiedantibodies, antibody fragments, and probes thereof.

To express the antibodies, or antibody portions of the invention, DNAsencoding partial or full-length light and heavy chains, obtained asdescribed above, are inserted into expression vectors such that thegenes are operatively linked to transcriptional and translationalcontrol sequences. Expression vectors include plasmids, retroviruses,cosmids, YACs, EBV derived episomes, and the like. The antibody gene isligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector. In a preferred embodiment, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by standard methods (e.g., ligation of complementary restrictionsites on the antibody gene fragment and vector, or blunt end ligation ifno restriction sites are present).

A convenient vector is one that encodes a functionally complete human CHor CL immunoglobulin sequence, with appropriate restriction sitesengineered so that any VH or VL sequence can be easily inserted andexpressed, as described above. In such vectors, splicing usually occursbetween the splice donor site in the inserted J region and the spliceacceptor site preceding the human C region, and also at the spliceregions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The recombinant expression vector can also encodea signal peptide that facilitates secretion of the antibody chain from ahost cell. The antibody chain gene may be cloned into the vector suchthat the signal peptide is linked in-frame to the amino terminus of theantibody chain gene. The signal peptide can be an immunoglobulin signalpeptide or a heterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. It will beappreciated by those skilled in the art that the design of theexpression vector, including the selection of regulatory sequences maydepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. Preferred regulatorysequences for mammalian host cell expression include viral elements thatdirect high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from retroviral LTRs, cytomegalovirus(CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (suchas the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus majorlate promoter (AdMLP)), polyoma and strong mammalian promoters such asnative immunoglobulin and actin promoters. For further description ofviral regulatory elements, and sequences thereof, see e.g., U.S. Pat.No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al. andU.S. Pat. No. 4,968,615 by Schaffner et al.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

Non-Hybridoma Host Cells and Methods of Recombinantly Producing Protein

Nucleic acid molecules encoding the heavy chain or an antigen-bindingportion thereof and/or the light chain or an antigen-binding portionthereof of an anti-IGF-IR antibody, and vectors comprising these nucleicacid molecules, can be used for transformation of a suitable mammalianhost cell. Transformation can be by any known method for introducingpolynucleotides into a host cell. Methods for introduction ofheterologous polynucleotides into mammalian cells are well known in theart and include dextran-mediated transfection, calcium phosphateprecipitation, polybrene-mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,biolistic injection and direct microinjection of the DNA into nuclei. Inaddition, nucleic acid molecules may be introduced into mammalian cellsby viral vectors. Methods of transforming cells are well known in theart. See, e.g., U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and4,959,455 (which patents are hereby incorporated herein by reference).

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC). These include, inter alia,Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, babyhamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells,and a number of other cell lines. Mammalian host cells include human,mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells.Cell lines of particular preference are selected through determiningwhich cell lines have high expression levels. Other cell lines that maybe used are insect cell lines, such as Sf9 cells, amphibian cells,bacterial cells, plant cells and fungal cells. When recombinantexpression vectors encoding the heavy chain or antigen-binding portionthereof, the light chain and/or antigen-binding portion thereof areintroduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or, more preferably,secretion of the antibody into the culture medium in which the hostcells are grown. Antibodies can be recovered from the culture mediumusing standard protein purification methods.

Further, expression of antibodies of the invention (or other moietiestherefrom) from production cell lines can be enhanced using a number ofknown techniques. For example, the glutamine synthetase gene expressionsystem (the GS system) is a common approach for enhancing expressionunder certain conditions. The GS system is discussed in whole or part inconnection with European Patent Nos. 0 216 846, 0 256 055, and 0 323 997and European Patent Application No. 89303964.4.

It is likely that antibodies expressed by different cell lines or intransgenic animals will have different glycosylation from each other.However, all antibodies encoded by the nucleic acid molecules providedherein, or comprising the amino acid sequences provided herein are partof the instant invention, regardless of the glycosylation of theantibodies.

Transgenic Animals

The invention also provides transgenic non-human animals comprising oneor more nucleic acid molecules of the invention that may be used toproduce antibodies of the invention. Antibodies can be produced in andrecovered from tissue or bodily fluids, such as milk, blood or urine, ofgoats, cows, horses, pigs, rats, mice, rabbits, hamsters or othermammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750,172, and5,741,957. As described above, non-human transgenic animals thatcomprise human immunoglobulin loci can be produced by immunizing withIGF-IR or a portion thereof.

In another embodiment, non-human transgenic animals are produced byintroducing one or more nucleic acid molecules of the invention into theanimal by standard transgenic techniques. See Hogan, supra. Thetransgenic cells used for making the transgenic animal can be embryonicstem cells or somatic cells. The transgenic non-human organisms can bechimeric, nonchimeric heterozygotes, and nonchimeric homozygotes. See,e.g., Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual2ed., Cold Spring Harbor Press (1999); Jackson et al., Mose Genetics andTransgenics: A Practical Approach, Oxford University Press (2000); andPinkert, Transgenic Animal Technology: A Laboratory Handbook, AcademicPress (1999). In another embodiment, the transgenic non-human organismsmay have a targeted disruption and replacement that encodes a heavychain and/or a light chain of interest. In a preferred embodiment, thetransgenic animals comprise and express nucleic acid molecules encodingheavy and light chains that bind specifically to IGF-IR, preferablyhuman IGF-IR. In another embodiment, the transgenic animals comprisenucleic acid molecules encoding a modified antibody such as asingle-chain antibody, a chimeric antibody or a humanized antibody. Theanti-IGF-IR antibodies may be made in any transgenic animal. In apreferred embodiment, the non-human animals are mice, rats, sheep, pigs,goats, cattle or horses. The non-human transgenic animal expresses saidencoded polypeptides in blood, milk, urine, saliva, tears, mucus andother bodily fluids.

Phage Display Libraries

The invention provides a method for producing an anti-IGF-IR antibody orantigen-binding portion thereof comprising the steps of synthesizing alibrary of human antibodies on phage, screening the library with aIGF-IR or a portion thereof, isolating phage that bind IGF-IR, andobtaining the antibody from the phage. One method to prepare the libraryof antibodies comprises the steps of immunizing a non-human host animalcomprising a human immunoglobulin locus with IGF-IR or an antigenicportion thereof to create an immune response, extracting cells from thehost animal the cells that are responsible for production of antibodies;isolating RNA from the extracted cells, reverse transcribing the RNA toproduce cDNA, amplifying the cDNA using a primer, and inserting the cDNAinto phage display vector such that antibodies are expressed on thephage. Recombinant anti-IGF-IR antibodies of the invention may beobtained in this way.

Recombinant anti-IGF-IR human antibodies of the invention in addition tothe anti-IGF-IR antibodies disclosed herein can be isolated by screeningof a recombinant combinatorial antibody library, preferably a scFv phagedisplay library, prepared using human VL and VH cDNAs prepared from mRNAderived from human lymphocytes. Methodologies for preparing andscreening such libraries are known in the art. There are commerciallyavailable kits for generating phage display libraries (e.g., thePharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01; andthe Stratagene SurfZAP™ phage display kit, catalog no. 240612). Thereare also other methods and reagents that can be used in generating andscreening antibody display libraries (see, e.g., Ladner et al. U.S. Pat.No. 5,223,409; Kang et al. PCT Publication No. WO 92/18619; Dower et al.PCT Publication No. WO 91/17271; Winter et al. PCT Publication No. WO92/20791; Markland et al. PCT Publication No. WO 92/15679; Breitling etal. PCT Publication No. WO 93/01288; McCafferty et al. PCT PublicationNo. WO 92/01047; Garrard et al. PCT Publication No. WO 92/09690; Fuchset al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;McCafferty et al., Nature (1990) 348:552-554; Griffiths et al. (1993)EMBO J 12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896;Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) Proc.Natl. Acad. Sci. USA 89:3576-3580; Garrad et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; andBarbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982.

In a preferred embodiment, to isolate human anti-IGF-IR antibodies withthe desired characteristics, a human anti-IGF-IR antibody as describedherein is first used to select human heavy and light chain sequenceshaving similar binding activity toward IGF-IR, using the epitopeimprinting methods described in Hoogenboom et al., PCT Publication No.WO 93/06213. The antibody libraries used in this method are preferablyscFv libraries prepared and screened as described in McCafferty et al.,PCT Publication No. WO 92/01047, McCafferty et al., Nature (1990)348:552-554; and Griffiths et al., (1993) EMBO J 12:725-734. The scFvantibody libraries preferably are screened using human IGF-IR as theantigen.

Once initial human VL and VH segments are selected, “mix and match”experiments, in which different pairs of the initially selected VL andVH segments are screened for IGF-IR binding, are performed to selectpreferred VL/VH pair combinations. Additionally, to further improve thequality of the antibody, the VL and VH segments of the preferred VL/VHpair(s) can be randomly mutated, preferably within the CDR3 region of VHand/or VL, in a process analogous to the in vivo somatic mutationprocess responsible for affinity maturation of antibodies during anatural immune response. This in vitro affinity maturation can beaccomplished by amplifying VH and VL regions using PCR primerscomplimentary to the VH CDR3 or VL CDR3, respectively, which primershave been “spiked” with a random mixture of the four nucleotide bases atcertain positions such that the resultant PCR products encode VH and VLsegments into which random mutations have been introduced into the VHand/or VL CDR3 regions. These randomly mutated VH and VL segments can berescreened for binding to IGF-IR.

Following screening and isolation of an anti-IGF-IR antibody of theinvention from a recombinant immunoglobulin display library, nucleicacid encoding the selected antibody can be recovered from the displaypackage (e.g., from the phage genome) and subcloned into otherexpression vectors by standard recombinant DNA techniques. If desired,the nucleic acid can be further manipulated to create other antibodyforms of the invention, as described below. To express a recombinanthuman antibody isolated by screening of a combinatorial library, the DNAencoding the antibody is cloned into a recombinant expression vector andintroduced into a mammalian host cells, as described above.

Class Switching

Another aspect of the instant invention is to provide a mechanism bywhich the class of an anti-IGF-IR antibody may be switched with another.In one aspect of the invention, a nucleic acid molecule encoding VL orVH is isolated using methods well-known in the art such that it does notinclude any nucleic acid sequences encoding CL or CH. The nucleic acidmolecule encoding VL or VH are then operatively linked to a nucleic acidsequence encoding a CL or CH from a different class of immunoglobulinmolecule. This may be achieved using a vector or nucleic acid moleculethat comprises a CL or CH chain, as described above. For example, ananti-IGF-IR antibody that was originally IgM may be class switched to anIgG. Further, the class switching may be used to convert one IgGsubclass to another, e.g., from IgG1 to IgG2. A preferred method forproducing an antibody of the invention comprising a desired isotypescomprises the steps of isolating a nucleic acid encoding the heavy chainof an anti-IGF-IR antibody and a nucleic acid encoding the light chainof an anti-IGF-IR antibody, obtaining the variable region of the heavychain, ligating the variable region of the heavy chain with the constantdomain of a heavy chain of the desired isotype, expressing the lightchain and the ligated heavy chain in a cell, and collecting theanti-IGF-IR antibody with the desired isotype.

Antibody Derivatives

One may use the nucleic acid molecules described above to generateantibody derivatives using techniques and methods known to one ofordinary skill in the art.

Humanized Antibodies

As was discussed above in connection with human antibody generation,there are advantages to producing antibodies with reducedimmunogenicity. This can be accomplished to some extent using techniquesof humanization and display techniques using appropriate libraries. Itwill be appreciated that murine antibodies or antibodies from otherspecies can be humanized or primatized using techniques well known inthe art. See e.g., Winter and Harris Immunol Today 14:43-46 (1993) andWright et al. Grit. Reviews in Immunol. 12125-168 (1992). The antibodyof interest may be engineered by recombinant DNA techniques tosubstitute the CH1, CH2, CH3, hinge domains, and/or the framework domainwith the corresponding human sequence (see WO 92/02190 and U.S. Pat.Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and5,777,085). In a preferred embodiment, the anti-IGF-IR antibody can behumanized by substituting the CH1, CH2, CH3, hinge domains, and/or theframework domain with the corresponding human sequence while maintainingall of the CDRS of the heavy chain, the light chain or both the heavyand light chains.

Mutated Antibodies

In another embodiment, the nucleic acid molecules, vectors and hostcells may be used to make mutated anti-IGF-IR antibodies. The antibodiesmay be mutated in the variable domains of the heavy and/or light chainsto alter a binding property of the antibody. For example, a mutation maybe made in one or more of the CDR regions to increase or decrease theK_(d) of the antibody for IGF-IR, to increase or decrease K_(off), or toalter the binding specificity of the antibody. Techniques insite-directed mutagenesis are well-known in the art. See, e.g., Sambrooket al. and Ausubel et al., supra. In a preferred embodiment, mutationsare made at an amino acid residue that is known to be changed comparedto germline in a variable region of an anti-IGF-IR antibody. In a morepreferred embodiment, one or more mutations are made at an amino acidresidue that is known to be changed compared to the germline in avariable region or CDR region of one of the anti-IGF-IR antibodies2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, 4.17.3 or 6.1.1. In anotherembodiment, one or more mutations are made at an amino acid residue thatis known to be changed compared to the germline in a variable region orCDR region whose amino acid sequence is presented in SEQ ID NOS: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22 or 24, or whose nucleic acid sequenceis presented in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23.In another embodiment, the nucleic acid molecules are mutated in one ormore of the framework regions. A mutation may be made in a frameworkregion or constant domain to increase the half-life of the anti-IGF-IRantibody. See, e.g., WO 00/09560, published Feb. 24, 2000, hereinincorporated by reference. In one embodiment, there may be one, three orfive point mutations and no more than ten point mutations. A mutation ina framework region or constant domain may also be made to alter theimmunogenicity of the antibody, to provide a site for covalent ornon-covalent binding to another molecule, or to alter such properties ascomplement fixation. Mutations may be made in each of the frameworkregions, the constant domain and the variable regions in a singlemutated antibody. Alternatively, mutations may be made in only one ofthe framework regions, the variable regions or the constant domain in asingle mutated antibody.

In one embodiment, there are no greater than ten amino acid changes ineither the VH or VL regions of the mutated anti-IGF-IR antibody comparedto the anti-IGF-IR antibody prior to mutation. In a more preferredembodiment, there is no more than five amino acid changes in either theVH or VL regions of the mutated anti-IGF-IR antibody, more preferably nomore than three amino acid changes. In another embodiment, there are nomore than fifteen amino acid changes in the constant domains, morepreferably, no more than ten amino acid changes, even more preferably,no more than five amino acid changes.

Modified Antibodies

In another embodiment, a fusion antibody or immunoadhesin may be madewhich comprises all or a portion of an anti-IGF-IR antibody linked toanother polypeptide. In a preferred embodiment, only the variableregions of the anti-IGF-IR antibody are linked to the polypeptide. Inanother preferred embodiment, the VH domain of an anti-IGF-IR antibodyare linked to a first polypeptide, while the VL domain of an anti-IGF-IRantibody are linked to a second polypeptide that associates with thefirst polypeptide in a manner in which the VH and VL domains caninteract with one another to form an antibody binding site. In anotherpreferred embodiment, the VH domain is separated from the VL domain by alinker such that the VH and VL domains can interact with one another(see below under Single Chain Antibodies). The VH-linker-VL antibody isthen linked to the polypeptide of interest. The fusion antibody isuseful to directing a polypeptide to an IGF-IR-expressing cell ortissue. The polypeptide may be a therapeutic agent, such as a toxin,growth factor or other regulatory protein, or may be a diagnostic agent,such as an enzyme that may be easily visualized, such as horseradishperoxidase. In addition, fusion antibodies can be created in which two(or more) single-chain antibodies are linked to one another. This isuseful if one wants to create a divalent or polyvalent antibody on asingle polypeptide chain, or if one wants to create a bispecificantibody.

To create a single chain antibody, (scFv) the VH- and VL-encoding DNAfragments are operatively linked to another fragment encoding a flexiblelinker, e.g., encoding the amino acid sequence (Gly₄-Ser)₃ (SEQ ID NO:60), such that the VH and VL sequences can be expressed as a contiguoussingle-chain protein, with the VL and VH regions joined by the flexiblelinker (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al.(1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al.,Nature (1990) 348:552-554). The single chain antibody may be monovalent,if only a single VH and VL are used, bivalent, if two VH and VL areused, or polyvalent, if more than two VH and VL are used.

In another embodiment, other modified antibodies may be prepared usinganti-IGF-IR-encoding nucleic acid molecules. For instance, “Kappabodies” (Ill et al., Protein Eng 10: 949-57 (1997)), “Minibodies”(Martin et al., EMBO J. 13: 5303-9 (1994)), “Diabodies” (Holliger etal., PNAS USA 90: 6444-6448 (1993)), or “Janusins” (Traunecker et al.,EMBO J 10: 3655-3659 (1991) and Traunecker et al. “Janusin: newmolecular design for bispecific reagents” Int J Cancer Suppl 7:51-52(1992)) may be prepared using standard molecular biological techniquesfollowing the teachings of the specification.

In another aspect, chimeric and bispecific antibodies can be generated.A chimeric antibody may be made that comprises CDRs and frameworkregions from different antibodies. In a preferred embodiment, the CDRsof the chimeric antibody comprises all of the CDRs of the variableregion of a light chain or heavy chain of an anti-IGF-IR antibody, whilethe framework regions are derived from one or more different antibodies.In a more preferred embodiment, the CDRs of the chimeric antibodycomprise all of the CDRs of the variable regions of the light chain andthe heavy chain of an anti-IGF-IR antibody. The framework regions may befrom another species and may, in a preferred embodiment, be humanized.Alternatively, the framework regions may be from another human antibody.

A bispecific antibody can be generated that binds specifically to IGF-IRthrough one binding domain and to a second molecule through a secondbinding domain. The bispecific antibody can be produced throughrecombinant molecular biological techniques, or may be physicallyconjugated together. In addition, a single chain antibody containingmore than one VH and VL may be generated that binds specifically toIGF-IR and to another molecule. Such bispecific antibodies can begenerated using techniques that are well known for example, inconnection with (i) and (ii) see e.g., Fanger et al. Immunol Methods 4:72-81 (1994) and Wright and Harris, supra. and in connection with (iii)see e.g., Traunecker et al. Int. J. Cancer (Suppl.) 7: 51-52 (1992). Ina preferred embodiment, the bispecific antibody binds to IGF-IR and toanother molecule expressed at high level on cancer or tumor cells. In amore preferred embodiment, the other molecule is erbB2 receptor, VEGF,CD20 or EGF-R.

In a embodiment, the modified antibodies described above are preparedusing one or more of the variable regions or one or more CDR regionsfrom one of the antibodies selected from 2.12.1, 2.13.2, 2.14.3, 3.1.1,4.9.2, 4.17.3 or 6.1.1. In another embodiment, the modified antibodiesare prepared using one or more of the variable regions or one or moreCDR regions whose amino acid sequence is presented in SEQ ID NOS: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22 or 24, or whose nucleic acid sequenceis presented in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23.

Derivatized and Labeled Antibodies

An antibody or antibody portion of the invention can be derivatized orlinked to another molecule (e.g., another peptide or protein). Ingeneral, the antibodies or portion thereof is derivatized such that theIGF-IR binding is not affected adversely by the derivatization orlabeling. Accordingly, the antibodies and antibody portions of theinvention are intended to include both intact and modified forms of thehuman anti-IGF-IR antibodies described herein. For example, an antibodyor antibody portion of the invention can be functionally linked (bychemical coupling, genetic fusion, noncovalent association or otherwise)to one or more other molecular entities, such as another antibody (e.g.,a bispecific antibody or a diabody), a detection agent, a cytotoxicagent, a pharmaceutical agent, and/or a protein or peptide that canmediate associate of the antibody or antibody portion with anothermolecule (such as a streptavidin core region or a polyhistidine tag).

One type of derivatized antibody is produced by crosslinking two or moreantibodies (of the same type or of different types, e.g., to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

Another type of derivatized antibody is a labeled antibody. Usefuldetection agents with which an antibody or antibody portion of theinvention may be derivatized include fluorescent compounds, includingfluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors and the like. An antibody may also be labeled with enzymesthat are useful for detection, such as horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase andthe like. When an antibody is labeled with a detectable enzyme, it isdetected by adding additional reagents that the enzyme uses to produce areaction product that can be discerned. For example, when the agenthorseradish peroxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be labeled with biotin, and detectedthrough indirect measurement of avidin or streptavidin binding. Anantibody may be labeled with a magnetic agent, such as gadolinium. Anantibody may also be labeled with a predetermined polypeptide epitopesrecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags). In some embodiments, labels are attached by spacer arms ofvarious lengths to reduce potential steric hindrance.

An anti-IGF-IR antibody may also be labeled with a radiolabeled aminoacid. The radiolabel may be used for both diagnostic and therapeuticpurposes. For instance, the radiolabel may be used to detectIGF-IR-expressing tumors by x-ray or other diagnostic techniques.Further, the radiolabel may be used therapeutically as a toxin forcancerous cells or tumors. Examples of labels for polypeptides include,but are not limited to, the following radioisotopes or radionuclides—³H,¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I.

An anti-IGF-IR antibody may also be derivatized with a chemical groupsuch as polyethylene glycol (PEG), a methyl or ethyl group, or acarbohydrate group. These groups may be useful to improve the biologicalcharacteristics of the antibody, e.g., to increase serum half-life or toincrease tissue binding.

Pharmaceutical Compositions and Kits

The invention also relates to a pharmaceutical composition for thetreatment of a hyperproliferative disorder in a mammal which comprises atherapeutically effective amount of a compound of the invention and apharmaceutically acceptable carrier. In one embodiment, saidpharmaceutical composition is for the treatment of cancer such as brain,lung, squamous cell, bladder, gastric, pancreatic, breast, head, neck,renal, kidney, ovarian, prostate, colorectal, esophageal, gynecologicalor thyroid cancer. In another embodiment, said pharmaceuticalcomposition relates to non-cancerous hyperproliferative disorders suchas, without limitation, restenosis after angioplasty and psoriasis. Inanother embodiment, the invention relates to pharmaceutical compositionsfor the treatment of a mammal that requires activation of IGF-IR,wherein the pharmaceutical composition comprises a therapeuticallyeffective amount of an activating antibody of the invention and apharmaceutically acceptable carrier. Pharmaceutical compositionscomprising activating antibodies may be used to treat animals that lacksufficient IGF-I or IGF-II, or may be used to treat osteoporosis,frailty or disorders in which the mammal secretes too little activegrowth hormone or is unable to respond to growth hormone.

The anti-IGF-IR antibodies of the invention can be incorporated intopharmaceutical compositions suitable for administration to a subject.Typically, the pharmaceutical composition comprises an antibody of theinvention and a pharmaceutically acceptable carrier. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. Examples of pharmaceutically acceptablecarriers include one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Pharmaceuticallyacceptable substances such as wetting or minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of the antibodyor antibody portion.

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans with other antibodies. Thepreferred mode of administration is parenteral (e.g., intravenous,subcutaneous, intraperitoneal, intramuscular). In a preferredembodiment, the antibody is administered by intravenous infusion orinjection. In another preferred embodiment, the antibody is administeredby intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the anti-IGF-IR antibody inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

The antibodies of the present invention can be administered by a varietyof methods known in the art, although for many therapeutic applications,the preferred route/mode of administration is intraperitoneal,subcutaneous, intramuscular, intravenous or infusion. As will beappreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results. In oneembodiment, the antibodies of the present invention can be administeredas a single dose or may be administered as multiple doses.

In certain embodiments, the active compound may be prepared with acarrier that will protect the compound against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978. In certain embodiments, the anti-IGF-IR ofthe invention may be orally administered, for example, with an inertdiluent or an assimilable edible carrier. The compound (and otheringredients, if desired) may also be enclosed in a hard or soft shellgelatin capsule, compressed into tablets, or incorporated directly intothe subject's diet. For oral therapeutic administration, the compoundsmay be incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. To administer a compound of the inventionby other than parenteral administration, it may be necessary to coat thecompound with, or co-administer the compound with, a material to preventits inactivation.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, an anti-IGF-IR of the invention iscoformulated with and/or coadministered with one or more additionaltherapeutic agents, such as a chemotherapeutic agent, an antineoplasticagent or an anti-tumor agent. For example, an anti-IGF-IR antibody maybe coformulated and/or coadministered with one or more additionaltherapeutic agents. These agents include, without limitation, antibodiesthat bind other targets (e.g., antibodies that bind one or more growthfactors or cytokines, their cell surface receptors or IGF-I), IGF-Ibinding proteins, antineoplastic agents, chemotherapeutic agents,anti-tumor agents, antisense oligonucleotides against IGF-IR or IGF-I,peptide analogues that block IGF-IR activation, soluble IGF-IR, and/orone or more chemical agents that inhibit IGF-I production or activity,which are known in the art, e.g., octreotide. For a pharmaceuticalcomposition comprising an activating antibody, the anti-IGF-IR antibodymay be formulated with a factor that increases cell proliferation orprevents apoptosis. Such factors include growth factors such as IGF-I,and/or analogues of IGF-I that activate IGF-IR. Such combinationtherapies may require lower dosages of the anti-IGF-IR antibody as wellas the co-administered agents, thus avoiding possible toxicities orcomplications associated with the various monotherapies. In oneembodiment, the antibody and one or more additional therapeutic agent.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody portion of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the antibodyor antibody portion may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of theantibody or antibody portion to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. Pharmaceuticalcomposition comprising the antibody or comprising a combination therapycomprising the antibody and one or more additional therapeutic agentsmay be formulated for single or multiple doses. It is especiallyadvantageous to formulate parenteral compositions in dosage unit formfor ease of administration and uniformity of dosage. Dosage unit form asused herein refers to physically discrete units suited as unitarydosages for the mammalian subjects to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals. A particularly useful formulation is 5 mg/mlanti-IGF-IR antibody in a buffer of 20 mM sodium citrate, pH 5.5, 140 mMNaCl, and 0.2 mg/ml polysorbate 80.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antibody portion ofthe invention is 0.1-100 mg/kg, more preferably 0.5-50 mg/kg, morepreferably 1-20 mg/kg, and even more preferably 1-10 mg/kg. It is to benoted that dosage values may vary with the type and severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition. In one embodiment, the therapeutically or prophylacticallyeffective amount of an antibody or antigen-binding portion thereof isadministered along with one or more additional therapeutic agents.

In another aspect, the invention relates to administration of ananti-IGF-IR antibody for the treatment of cancer in a dose of less than300 mg per month.

Another aspect of the present invention provides kits comprising theanti-IGF-IR antibodies and the pharmaceutical compositions comprisingthese antibodies. A kit may include, in addition to the antibody orpharmaceutical composition, diagnostic or therapeutic agents. A kit mayalso include instructions for use in a diagnostic or therapeutic method.In a preferred embodiment, the kit includes the antibody or apharmaceutical composition thereof and a diagnostic agent that can beused in a method described below. In another preferred embodiment, thekit includes the antibody or a pharmaceutical composition thereof andone or more therapeutic agents, such as an additional antineoplasticagent, anti-tumor agent or chemotherapeutic agent, that can be used in amethod described below.

This invention also relates to pharmaceutical compositions forinhibiting abnormal cell growth in a mammal which comprise an amount ofa compound of the invention in combination with an amount of achemotherapeutic agent, wherein the amounts of the compound, salt,solvate, or prodrug, and of the chemotherapeutic agent are togethereffective in inhibiting abnormal cell growth. Many chemotherapeuticagents are presently known in the art. In one embodiment, thechemotherapeutic agents is selected from the group consisting of mitoticinhibitors, alkylating agents, anti-metabolites, intercalatingantibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes,topoisomerase inhibitors, anti-survival agents, biological responsemodifiers, anti-hormones, e.g. anti-androgens, and anti-angiogenesisagents.

Anti-angiogenesis agents, such as MMP-2 (matrix-metalloproteinase 2)inhibitors, MMP-9 (matrix-metalloproteinase 9) inhibitors, and COX-II(cyclooxygenase II) inhibitors, can be used in conjunction with acompound of the invention. Examples of useful COX-II inhibitors includeCELEBREX™ (alecoxib), valdecoxib, and rofecoxib. Examples of usefulmatrix metalloproteinase inhibitors are described in WO 96/33172(published Oct. 24, 1996), WO 96/27583 (published Mar. 7, 1996),European Patent Application No. 97304971.1 (filed Jul. 8, 1997),European Patent Application No. 99308617.2 (filed Oct. 29, 1999), WO98/07697 (published Feb. 26, 1998), WO 98/03516 (published Jan. 29,1998), WO 98/34918 (published Aug. 13, 1998), WO 98/34915 (publishedAug. 13, 1998), WO 98/33768 (published Aug. 6, 1998), WO 98/30566(published Jul. 16, 1998), European Patent Publication 606,046(published Jul. 13, 1994), European Patent Publication 931,788(published Jul. 28, 1999), WO 90/05719 (published May 31, 1990), WO99/52910 (published Oct. 21, 1999), WO 99/52889 (published Oct. 21,1999), WO 99/29667 (published Jun. 17, 1999), PCT InternationalApplication No. PCT/IB98/01113 (filed Jul. 21, 1998), European PatentApplication No. 99302232.1 (filed Mar. 25, 1999), Great Britain patentapplication number 9912961.1 (filed Jun. 3, 1999), U.S. ProvisionalApplication No. 60/148,464 (filed Aug. 12, 1999), U.S. Pat. No.5,863,949 (issued Jan. 26, 1999), U.S. Pat. No. 5,861,510 (issued Jan.19, 1999), and European Patent Publication 780,386 (published Jun. 25,1997), all of which are incorporated herein in their entireties byreference. Preferred MMP inhibitors are those that do not demonstratearthralgia. More preferred, are those that selectively inhibit MMP-2and/or MMP-9 relative to the other matrix-metalloproteinases (i.e.MMP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12,and MMP-13). Some specific examples of MMP inhibitors useful in thepresent invention are AG-3340, RO 32-3555, RS 13-0830, and the compoundsrecited in the following list:3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclopentyl)-amino]-propionicacid;3-exo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3-carboxylicacid hydroxyamide; (2R,3R)1-[4-(2-chloro-4-fluoro-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylicacid hydroxyamide;4-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylicacid hydroxyamide;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-cyclobutyl)-amino]-propionicacid;4-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylicacid hydroxyamide; (R)3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-3-carboxylicacid hydroxyamide; (2R,3R)1-[4-(4-fluoro-2-methyl-benzyloxy)-benzenesulfonyl]-3-hydroxy-3-methyl-piperidine-2-carboxylicacid hydroxyamide;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(1-hydroxycarbamoyl-1-methyl-ethyl)-amino]-propionicacid;3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(4-hydroxycarbamoyl-tetrahydro-pyran-4-yl)-amino]-propionicacid;3-exo-3-[4-(4-chloro-phenoxy)-benzenesulfonylamino]-8-oxa-icyclo[3.2.1]octane-3-carboxylicacid hydroxyamide;3-endo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-icyclo[3.2.1]octane-3-carboxylicacid hydroxyamide; and (R)3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-tetrahydro-furan-3-carboxylicacid hydroxyamide; and pharmaceutically acceptable salts and solvates ofsaid compounds.

A compound of the invention can also be used with signal transductioninhibitors, such as agents that can inhibit EGF-R (epidermal growthfactor receptor) responses, such as EGF-R antibodies, EGF antibodies,and molecules that are EGF-R inhibitors; VEGF (vascular endothelialgrowth factor) inhibitors, such as VEGF receptors and molecules that caninhibit VEGF; and erbB2 receptor inhibitors, such as organic moleculesor antibodies that bind to the erbB2 receptor, for example, HERCEPTIN™(Genentech, Inc.). EGF-R inhibitors are described in, for example in WO95/19970 (published Jul. 27, 1995), WO 98/14451 (published Apr. 9,1998), WO 98/02434 (published Jan. 22, 1998), and U.S. Pat. No.5,747,498 (issued May 5, 1998), and such substances can be used in thepresent invention as described herein. EGFR-inhibiting agents include,but are not limited to, the monoclonal antibodies C225 and anti-EGFR22Mab (ImClone Systems Incorporated), ABX-EGF (Abgenix/Cell Genesys),EMD-7200 (Merck KgaA), EMD-5590 (Merck KgaA), MDX-447/H-477 (MedarexInc. and Merck KgaA), and the compounds ZD-1834, ZD-1838 and ZD-1839(AstraZeneca), PKI-166 (Novartis), PKI-166/CGP-75166 (Novartis), PTK 787(Novartis), CP 701 (Cephalon), leflunomide (Pharmacia/Sugen), CI-1033(Warner Lambert Parke Davis), CI-1033/PD 183,805 (Warner Lambert ParkeDavis), CL-387,785 (Wyeth-Ayerst), BBR-1611 (Boehringer MannheimGmbH/Roche), Naamidine A (Bristol Myers Squibb), RC-3940-II (Pharmacia),BIBX-1382 (Boehringer Ingelheim), OLX-103 (Merck & Co.), VRCTC-310(Ventech Research), EGF fusion toxin (Seragen Inc.), DAB-389(Seragen/Lilgand), ZM-252808 (Imperial Cancer Research Fund), RG-50864(INSERM), LFM-A12 (Parker Hughes Cancer Center), WHI-P97 (Parker HughesCancer Center), GW-282974 (Glaxo), KT-8391 (Kyowa Hakko) and EGF-RVaccine (York Medical/Centro de Immunologia Molecular (CIM)). These andother EGF-R-inhibiting agents can be used in the present invention.

VEGF inhibitors, for example SU-5416 and SU-6668 (Sugen Inc.), SH-268(Schering), and NX-1838 (NeXstar) can also be combined with the compoundof the present invention. VEGF inhibitors are described in, for examplein WO 99/24440 (published May 20, 1999), PCT International ApplicationPCT/IB99/00797 (filed May 3, 1999), in WO 95/21613 (published Aug. 17,1995), WO 99/61422 (published Dec. 2, 1999), U.S. Pat. No. 5,834,504(issued Nov. 10, 1998), WO 98/50356 (published Nov. 12, 1998), U.S. Pat.No. 5,883,113 (issued Mar. 16, 1999), U.S. Pat. No. 5,886,020 (issuedMar. 23, 1999), U.S. Pat. No. 5,792,783 (issued Aug. 11, 1998), WO99/10349 (published Mar. 4, 1999), WO 97/32856 (published Sep. 12,1997), WO 97/22596 (published Jun. 26, 1997), WO 98/54093 (publishedDec. 3, 1998), WO 98/02438 (published Jan. 22, 1998), WO 99/16755(published Apr. 8, 1999), and WO 98/02437 (published Jan. 22, 1998), allof which are incorporated herein in their entireties by reference. Otherexamples of some specific VEGF inhibitors useful in the presentinvention are IM862 (Cytran Inc.); anti-VEGF monoclonal antibody ofGenentech, Inc.; and angiozyme, a synthetic ribozyme from Ribozyme andChiron. These and other VEGF inhibitors can be used in the presentinvention as described herein.

ErbB2 receptor inhibitors, such as GW-282974 (Glaxo Wellcome plc), andthe monoclonal antibodies AR-209 (Aronex Pharmaceuticals Inc.) and 2B-1(Chiron), can furthermore be combined with the compound of theinvention, for example those indicated in WO 98/02434 (published Jan.22, 1998), WO 99/35146 (published Jul. 15, 1999), WO 99/35132 (publishedJul. 15, 1999), WO 98/02437 (published Jan. 22, 1998), WO 97/13760(published Apr. 17, 1997), WO 95/19970 (published Jul. 27, 1995), U.S.Pat. No. 5,587,458 (issued Dec. 24, 1996), and U.S. Pat. No. 5,877,305(issued Mar. 2, 1999), which are all hereby incorporated herein in theirentireties by reference. ErbB2 receptor inhibitors useful in the presentinvention are also described in U.S. Provisional Application No.60/117,341, filed Jan. 27, 1999, and in U.S. Provisional Application No.60/117,346, filed Jan. 27, 1999, both of which are incorporated in theirentireties herein by reference. The erbB2 receptor inhibitor compoundsand substance described in the aforementioned PCT applications, U.S.patents, and U.S. provisional applications, as well as other compoundsand substances that inhibit the erbB2 receptor, can be used with thecompound of the present invention in accordance with the presentinvention.

Anti-survival agents include anti-IGF-IR antibodies and anti-integrinagents, such as anti-integrin antibodies.

Diagnostic Methods of Use

The anti-IGF-IR antibodies may be used to detect IGF-IR in a biologicalsample in vitro or in vivo. The anti-IGF-IR antibodies may be used in aconventional immunoassay, including, without limitation, an ELISA, anRIA, FACS, tissue immunohistochemistry, Western blot orimmunoprecipitation. The anti-IGF-IR antibodies of the invention may beused to detect IGF-IR from humans. In another embodiment, theanti-IGF-IR antibodies may be used to detect IGF-IR from Old Worldprimates such as cynomologous and rhesus monkeys, chimpanzees and apes.The invention provides a method for detecting anti-IGF-IR in abiological sample comprising contacting a biological sample with ananti-IGF-IR antibody of the invention and detecting the bound antibodybound to anti-IGF-IR, to detect the IGF-IR in the biological sample. Inone embodiment, the anti-IGF-IR antibody is directly labeled with adetectable label. In another embodiment, the anti-IGF-IR antibody (thefirst antibody) is unlabeled and a second antibody or other moleculethat can bind the anti-IGF-IR antibody is labeled. As is well known toone of skill in the art, a second antibody is chosen that is able tospecifically bind the specific species and class of the first antibody.For example, if the anti-IGF-IR antibody is a human IgG, then thesecondary antibody may be an anti-human-IgG. Other molecules that canbind to antibodies include, without limitation, Protein A and Protein G,both of which are available commercially, e.g., from Pierce Chemical Co.

Suitable labels for the antibody or secondary have been disclosed supra,and include various enzymes, prosthetic groups, fluorescent materials,luminescent materials, magnetic agents and radioactive materials.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; an example of amagnetic agent includes gadolinium; and examples of suitable radioactivematerial include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In an alternative embodiment, IGF-IR can be assayed in a biologicalsample by a competition immunoassay utilizing IGF-IR standards labeledwith a detectable substance and an unlabeled anti-IGF-IR antibody. Inthis assay, the biological sample, the labeled IGF-IR standards and theanti-IGF-IR antibody are combined and the amount of labeled IGF-IRstandard bound to the unlabeled antibody is determined. The amount ofIGF-IR in the biological sample is inversely proportional to the amountof labeled IGF-IR standard bound to the anti-IGF-IR antibody.

One may use the immunoassays disclosed above for a number of purposes.In one embodiment, the anti-IGF-IR antibodies may be used to detectIGF-IR in cells in cell culture. In a preferred embodiment, theanti-IGF-IR antibodies may be used to determine the level of tyrosinephosphorylation, tyrosine autophosphorylation of IGF-IR, and/or theamount of IGF-IR on the cell surface after treatment of the cells withvarious compounds. This method can be used to test compounds that may beused to activate or inhibit IGF-IR. In this method, one sample of cellsis treated with a test compound for a period of time while anothersample is left untreated. If tyrosine autophosphorylation is to bemeasured, the cells are lysed and tyrosine phosphorylation of the IGF-IRis measured using an immunoassay described above or as described inExample III, which uses an ELISA. If the total level of IGF-IR is to bemeasured, the cells are lysed and the total IGF-IR level is measuredusing one of the immunoassays described above.

A preferred immunoassay for determining IGF-IR tyrosine phosphorylationor for measuring total IGF-IR levels is an ELISA or Western blot. Ifonly the cell surface level of IGF-IR is to be measured, the cells arenot lysed, and the cell surface levels of IGF-IR are measured using oneof the immunoassays described above. A preferred immunoassay fordetermining cell surface levels of IGF-IR includes the steps of labelingthe cell surface proteins with a detectable label, such as biotin or¹²⁵I, immunoprecipitating the IGF-IR with an anti-IGF-IR antibody andthen detecting the labeled IGF-IR. Another preferred immunoassay fordetermining the localization of IGF-IR, e.g., cell surface levels, is byusing immunohistochemistry. Methods such as ELISA, RIA, Western blot,immunohistochemistry, cell surface labeling of integral membraneproteins and immunoprecipitation are well known in the art. See, e.g.,Harlow and Lane, supra. In addition, the immunoassays may be scaled upfor high throughput screening in order to test a large number ofcompounds for either activation or inhibition of IGF-IR.

The anti-IGF-IR antibodies of the invention may also be used todetermine the levels of IGF-IR in a tissue or in cells derived from thetissue. In a preferred embodiment, the tissue is a diseased tissue. In amore preferred embodiment, the tissue is a tumor or a biopsy thereof. Ina preferred embodiment of the method, a tissue or a biopsy thereof isexcised from a patient. The tissue or biopsy is then used in animmunoassay to determine, e.g., IGF-IR levels, cell surface levels ofIGF-IR, levels of tyrosine phosphorylation of IGF-IR, or localization ofIGF-IR by the methods discussed above. The method can be used todetermine if a tumor expresses IGF-IR at a high level.

The above-described diagnostic method can be used to determine whether atumor expresses high levels of IGF-IR, which may be indicative that thetumor will respond well to treatment with anti-IGF-IR antibody. Thediagnostic method may also be used to determine whether a tumor ispotentially cancerous, if it expresses high levels of IGF-IR, or benign,if it expresses low levels of IGF-IR. Further, the diagnostic method mayalso be used to determine whether treatment with anti-IGF-IR antibody(see below) is causing a tumor to express lower levels of IGF-IR and/orto express lower levels of tyrosine autophosphorylation, and thus can beused to determine whether the treatment is successful. In general, amethod to determine whether an anti-IGF-IR antibody decreases tyrosinephosphorylation comprises the steps of measuring the level of tyrosinephosphorylation in a cell or tissue of interest, incubating the cell ortissue with an anti-IGF-IR antibody or antigen-binding portion thereof,then re-measuring the level of tyrosine phosphorylation in the cell ortissue. The tyrosine phosphorylation of IGF-IR or of another protein(s)may be measured. The diagnostic method may also be used to determinewhether a tissue or cell is not expressing high enough levels of IGF-IRor high enough levels of activated IGF-IR, which may be the case forindividuals with dwarfism, osteoporosis or diabetes. A diagnosis thatlevels of IGF-IR or active IGF-IR are too low could be used fortreatment with activating anti-IGF-IR antibodies, IGF-I or othertherapeutic agents for increasing IGF-IR levels or activity.

The antibodies of the present invention may also be used in vivo tolocalize tissues and organs that express IGF-IR. In a preferredembodiment, the anti-IGF-IR antibodies can be used localizeIGF-IR-expressing tumors. The advantage of the anti-IGF-IR antibodies ofthe present invention is that they will not generate an immune responseupon administration. The method comprises the steps of administering ananti-IGF-IR antibody or a pharmaceutical composition thereof to apatient in need of such a diagnostic test and subjecting the patient toimaging analysis determine the location of the IGF-IR-expressingtissues. Imaging analysis is well known in the medical art, andincludes, without limitation, x-ray analysis, magnetic resonance imaging(MRI) or computed tomography (CE). In another embodiment of the method,a biopsy is obtained from the patient to determine whether the tissue ofinterest expresses IGF-IR rather than subjecting the patient to imaginganalysis. In a preferred embodiment, the anti-IGF-IR antibodies may belabeled with a detectable agent that can be imaged in a patient. Forexample, the antibody may be labeled with a contrast agent, such asbarium, which can be used for x-ray analysis, or a magnetic contrastagent, such as a gadolinium chelate, which can be used for MRI or CE.Other labeling agents include, without limitation, radioisotopes, suchas ⁹⁹Tc. In another embodiment, the anti-IGF-IR antibody will beunlabeled and will be imaged by administering a second antibody or othermolecule that is detectable and that can bind the anti-IGF-IR antibody.

Therapeutic Methods of Use

In another embodiment, the invention provides a method for inhibitingIGF-IR activity by administering an anti-IGF-IR antibody to a patient inneed thereof. Any of the types of antibodies described herein may beused therapeutically. In a preferred embodiment, the anti-IGF-IRantibody is a human, chimeric or humanized antibody. In anotherpreferred embodiment, the IGF-IR is human and the patient is a humanpatient. Alternatively, the patient may be a mammal that expresses anIGF-IR that the anti-IGF-IR antibody cross-reacts with. The antibody maybe administered to a non-human mammal expressing an IGF-IR with whichthe antibody cross-reacts (i.e. a primate, or a cynomologous or rhesusmonkey) for veterinary purposes or as an animal model of human disease.Such animal models may be useful for evaluating the therapeutic efficacyof antibodies of this invention.

As used herein, the term “a disorder in which IGF-IR activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of high levels of IGF-IR in a subject suffering fromthe disorder has been shown to be or is suspected of being eitherresponsible for the pathophysiology of the disorder or a factor thatcontributes to a worsening of the disorder. Accordingly, a disorder inwhich high levels of IGF-IR activity is detrimental is a disorder inwhich inhibition of IGF-IR activity is expected to alleviate thesymptoms and/or progression of the disorder. Such disorders may beevidenced, for example, by an increase in the levels of IGF-IR on thecell surface or in increased tyrosine autophosphorylation of IGF-IR inthe affected cells or tissues of a subject suffering from the disorder.The increase in IGF-IR levels may be detected, for example, using ananti-IGF-IR antibody as described above.

In a preferred embodiment, an anti-IGF-IR antibody may be administeredto a patient who has an IGF-IR-expressing tumor. A tumor may be a solidtumor or may be a non-solid tumor, such as a lymphoma. In a morepreferred embodiment, an anti-IGF-IR antibody may be administered to apatient who has an IGF-IR-expressing tumor that is cancerous. In an evenmore preferred embodiment, the anti-IGF-IR antibody is administered to apatient who has a tumor of the lung, breast, prostate or colon. In ahighly preferred embodiment, the method causes the tumor not to increasein weight or volume or to decrease in weight or volume. In anotherembodiment, the method causes the IGF-IR on the tumor to beinternalized. In a preferred embodiment, the antibody is selected from2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2, or 6.1.1, or comprises a heavychain, light chain or antigen-binding region thereof.

In another preferred embodiment, an anti-IGF-IR antibody may beadministered to a patient who expresses inappropriately high levels ofIGF-I. It is known in the art that high-level expression of IGF-I canlead to a variety of common cancers. In a more preferred embodiment, theanti-IGF-IR antibody is administered to a patient with prostate cancer,glioma or fibrosarcoma. In an even more preferred embodiment, the methodcauses the cancer to stop proliferating abnormally, or not to increasein weight or volume or to decrease in weight or volume.

In one embodiment, said method relates to the treatment of cancer suchas brain, squamous cell, bladder, gastric, pancreatic, breast, head,neck, esophageal, prostate, colorectal, lung, renal, kidney, ovarian,gynecological or thyroid cancer. Patients that can be treated with acompounds of the invention according to the methods of this inventioninclude, for example, patients that have been diagnosed as having lungcancer, bone cancer, pancreatic cancer, skin cancer, cancer of the headand neck, cutaneous or intraocular melanoma, uterine cancer, ovariancancer, rectal cancer, cancer of the anal region, stomach cancer, coloncancer, breast cancer, gynecologic tumors (e.g., uterine sarcomas,carcinoma of the fallopian tubes, carcinoma of the endometrium,carcinoma of the cervix, carcinoma of the vagina or carcinoma of thevulva), Hodgkin's disease, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system (e.g., cancer of the thyroid,parathyroid or adrenal glands), sarcomas of soft tissues, cancer of theurethra, cancer of the penis, prostate cancer, chronic or acuteleukemia, solid tumors of childhood, lymphocytic lymphomas, cancer ofthe bladder, cancer of the kidney or ureter (e.g., renal cell carcinoma,carcinoma of the renal pelvis), or neoplasms of the central nervoussystem (e.g., primary CNS lymphoma, spinal axis tumors, brain stemgliomas or pituitary adenomas).

The antibody may be administered once, but more preferably isadministered multiple times. The antibody may be administered from threetimes daily to once every six months. The administering may be on aschedule such as three times daily, twice daily, once daily, once everytwo days, once every three days, once weekly, once every two weeks, onceevery month, once every two months, once every three months and onceevery six months. The antibody may be administered via an oral, mucosal,buccal, intranasal, inhalable, intravenous, subcutaneous, intramuscular,parenteral, intratumor or topical route. The antibody may beadministered at a site distant from the site of the tumor. The antibodymay also be administered continuously via a minipump. The antibody maybe administered once, at least twice or for at least the period of timeuntil the condition is treated, palliated or cured. The antibodygenerally will be administered for as long as the tumor is presentprovided that the antibody causes the tumor or cancer to stop growing orto decrease in weight or volume. The antibody will generally beadministered as part of a pharmaceutical composition as described supra.The dosage of antibody will generally be in the range of 0.1-100 mg/kg,more preferably 0.5-50 mg/kg, more preferably 1-20 mg/kg, and even morepreferably 1-10 mg/kg. The serum concentration of the antibody may bemeasured by any method known in the art. See, e.g., Example XVII below.The antibody may also be administered prophylactically in order toprevent a cancer or tumor from occurring. This may be especially usefulin patients that have a “high normal” level of IGF-I because thesepatients have been shown to have a higher risk of developing commoncancers. See Rosen et al., supra.

In another aspect, the anti-IGF-IR antibody may be co-administered withother therapeutic agents, such as antineoplastic drugs or molecules, toa patient who has a hyperproliferative disorder, such as cancer or atumor. In one aspect, the invention relates to a method for thetreatment of the hyperproliferative disorder in a mammal comprisingadministering to said mammal a therapeutically effective amount of acompound of the invention in combination with an anti-tumor agentselected from the group consisting of, but not limited to, mitoticinhibitors, alkylating agents, anti-metabolites, intercalating agents,growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomeraseinhibitors, biological response modifiers, anti-hormones, kinaseinhibitors, matrix metalloprotease inhibitors, genetic therapeutics andanti-androgens. In a more preferred embodiment, the antibody may beadministered with an antineoplastic agent, such as adriamycin or taxol.In another preferred embodiment, the antibody or combination therapy isadministered along with radiotherapy, chemotherapy, photodynamictherapy, surgery or other immunotherapy. In yet another preferredembodiment, the antibody will be administered with another antibody. Forexample, the anti-IGF-IR antibody may be administered with an antibodyor other agent that is known to inhibit tumor or cancer cellproliferation, e.g., an antibody or agent that inhibits erbB2 receptor,EGF-R, CD20 or VEGF.

Co-administration of the antibody with an additional therapeutic agent(combination therapy) encompasses administering a pharmaceuticalcomposition comprising the anti-IGF-IR antibody and the additionaltherapeutic agent and administering two or more separate pharmaceuticalcompositions, one comprising the anti-IGF-IR antibody and the other(s)comprising the additional therapeutic agent(s). Further, althoughco-administration or combination therapy generally means that theantibody and additional therapeutic agents are administered at the sametime as one another, it also encompasses instances in which the antibodyand additional therapeutic agents are administered at different times.For instance, the antibody may be administered once every three days,while the additional therapeutic agent is administered once daily.Alternatively, the antibody may be administered prior to or subsequentto treatment of the disorder with the additional therapeutic agent.Similarly, administration of the anti-IGF-IR antibody may beadministered prior to or subsequent to other therapy, such asradiotherapy, chemotherapy, photodynamic therapy, surgery or otherimmunotherapy

The antibody and one or more additional therapeutic agents (thecombination therapy) may be administered once, twice or at least theperiod of time until the condition is treated, palliated or cured.Preferably, the combination therapy is administered multiple times. Thecombination therapy may be administered from three times daily to onceevery six months. The administering may be on a schedule such as threetimes daily, twice daily, once daily, once every two days, once everythree days, once weekly, once every two weeks, once every month, onceevery two months, once every three months and once every six months, ormay be administered continuously via a minipump. The combination therapymay be administered via an oral, mucosal, buccal, intranasal, inhalable,intravenous, subcutaneous, intramuscular, parenteral, intratumor ortopical route. The combination therapy may be administered at a sitedistant from the site of the tumor. The combination therapy generallywill be administered for as long as the tumor is present provided thatthe antibody causes the tumor or cancer to stop growing or to decreasein weight or volume.

In a still further embodiment, the anti-IGF-IR antibody is labeled witha radiolabel, an immunotoxin or a toxin, or is a fusion proteincomprising a toxic peptide. The anti-IGF-IR antibody or anti-IGF-IRantibody fusion protein directs the radiolabel, immunotoxin, toxin ortoxic peptide to the IGF-IR-expressing tumor or cancer cell. In apreferred embodiment, the radiolabel, immunotoxin, toxin or toxicpeptide is internalized after the anti-IGF-IR antibody binds to theIGF-IR on the surface of the tumor or cancer cell.

In another aspect, the anti-IGF-IR antibody may be used therapeuticallyto induce apoptosis of specific cells in a patient in need thereof. Inmany cases, the cells targeted for apoptosis are cancerous or tumorcells. Thus, in a preferred embodiment, the invention provides a methodof inducing apoptosis by administering a therapeutically effectiveamount of an anti-IGF-IR antibody to a patient in need thereof. In apreferred embodiment, the antibody is selected from 2.12.1, 2.13.2,2.14.3, 3.1.1, 4.9.2, or 6.1.1, or comprises a heavy chain, light chainor antigen-binding region thereof.

In another aspect, the anti-IGF-IR antibody may be used to treatnoncancerous states in which high levels of IGF-I and/or IGF-IR havebeen associated with the noncancerous state or disease. In oneembodiment, the method comprises the step of administering ananti-IGF-IR antibody to a patient who has a noncancerous pathologicalstate caused or exacerbated by high levels of IGF-I and/or IGF-IR levelsor activity. In a preferred embodiment, the noncancerous pathologicalstate is acromegaly, gigantism, psoriasis, atherosclerosis, smoothmuscle restenosis of blood vessels or inappropriate microvascularproliferation, such as that found as a complication of diabetes,especially of the eye. In a more preferred embodiment, the anti-IGF-IRantibody slows the progress of the noncancerous pathological state. In amore preferred embodiment, the anti-IGF-IR antibody stops or reverses,at least in part, the noncancerous pathological state.

In another aspect, the invention provides a method of administering anactivating anti-IGF-IR antibody to a patient in need thereof. In oneembodiment, the activating antibody or pharmaceutical composition isadministered to a patient in need thereof in an amount effective toincrease IGF-IR activity. In a more preferred embodiment, the activatingantibody is able to restore normal IGF-IR activity. In another preferredembodiment, the activating antibody may be administered to a patient whohas small stature, neuropathy, a decrease in muscle mass orosteoporosis. In another preferred embodiment, the activating antibodymay be administered with one or more other factors that increase cellproliferation, prevent apoptosis or increase IGF-IR activity. Suchfactors include growth factors such as IGF-I, and/or analogues of IGF-Ithat activate IGF-IR. In a preferred embodiment, the antibody isselected from 4.17.3, or comprises a heavy chain, light chain orantigen-binding portion thereof.

Gene Therapy

The nucleic acid molecules of the instant invention may be administeredto a patient in need thereof via gene therapy. The therapy may be eitherin vivo or ex vivo. In a preferred embodiment, nucleic acid moleculesencoding both a heavy chain and a light chain are administered to apatient. In a more preferred embodiment, the nucleic acid molecules areadministered such that they are stably integrated into the chromosome ofB cells because these cells are specialized for producing antibodies. Ina preferred embodiment, precursor B cells are transfected or infected exvivo and re-transplanted into a patient in need thereof. In anotherembodiment, precursor B cells or other cells are infected in vivo usinga virus known to infect the cell type of interest. Typical vectors usedfor gene therapy include liposomes, plasmids, or viral vectors, such asretroviruses, adenoviruses and adeno-associated viruses. After infectioneither in vivo or ex vivo, levels of antibody expression may bemonitored by taking a sample from the treated patient and using anyimmunoassay known in the art and discussed herein.

In a preferred embodiment, the gene therapy method comprises the stepsof administering an effective amount of an isolated nucleic acidmolecule encoding the heavy chain or the antigen-binding portion thereofof the human antibody or portion thereof and expressing the nucleic acidmolecule. In another embodiment, the gene therapy method comprises thesteps of administering an effective amount of an isolated nucleic acidmolecule encoding the light chain or the antigen-binding portion thereofof the human antibody or portion thereof and expressing the nucleic acidmolecule. In a more preferred method, the gene therapy method comprisesthe steps of administering an effective amount of an isolated nucleicacid molecule encoding the heavy chain or the antigen-binding portionthereof of the human antibody or portion thereof and an effective amountof an isolated nucleic acid molecule encoding the light chain or theantigen-binding portion thereof of the human antibody or portion thereofand expressing the nucleic acid molecules. The gene therapy method mayalso comprise the step of administering another anti-cancer agent, suchas taxol, tamoxifen, 5-FU, adriamycin or CP-358,774.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly and are not to be construed as limiting the scope of the inventionin any manner.

Example I Generation of Hybridomas Producing Anti-IGF-IR Antibody

Antibodies of the invention were prepared, selected, and assayed asfollows:

Immunization and Hybridoma Generation

Eight to ten week old XENOMICE™ were immunized intraperitoneally or intheir hind footpads with either the extracellular domain of human IGF-IR(10 μg/dose/mouse), or with 3T3-IGF-IR or 300.19-IGF-IR cells, which aretwo transfected cell lines that express human IGF-IR on their plasmamembranes (10×10⁶ cells/dose/mouse). This dose was repeated five toseven times over a three to eight week period. Four days before fusion,the mice received a final injection of the extracellular domain of humanIGF-IR in PBS. Spleen and lymph node lymphocytes from immunized micewere fused with the non-secretory myeloma P3-X63-Ag8.653 cell line andwere subjected to HAT selection as previously described (Galfre andMilstein, Methods Enzymol. 73:3-46, 1981). A panel of hybridomas allsecreting IGF-IR specific human IgG2a antibodies were recovered. Sevenhybridomas producing monoclonal antibodies specific for IGF-IR wereselected for further study and were designated 2.12.1, 2.13.2, 2.14.3,3.1.1, 4.9.2, 4.17.3 and 6.1.1.

Hybridomas 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2 and 4.17.3 weredeposited in the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110-2209, on Dec. 12, 2000 withthe following deposit numbers:

Hybridoma Deposit No. 2.12.1 PTA-2792 2.13.2 PTA-2788 2.14.3 PTA-27903.1.1 PTA-2791 4.9.2 PTA-2789 4.17.3 PTA-2793

Example II Determination of Affinity Constants (K_(d)) of Fully HumanAnti-IGF-IR Monoclonal Antibodies by BIAcore

We performed affinity measures of purified antibodies by surface plasmonresonance using the BIAcore 3000 instrument, following themanufacturer's protocols.

Protocol 1

To perform kinetic analyses, protein-A was immobilized on the sensorchipsurfaces of the BIAcore. The sensorchip was then used to capture theanti-IGF-IR antibodies of the present invention. Differentconcentrations of the extracellular domain of IGF-IR were injected onthe sensorchip and the binding and dissociation kinetics of theinteractions between the anti-IGF-IR antibodies and the extracellulardomain of IGF-IR were analyzed. The data were evaluated with global fitLangmuir 1:1, using baseline drift models available on the BIAevaluationsoftware provided by BIAcore.

Protocol 2

BIAcore measurements were performed essentially as described byFagerstam et al. “Detection of antigen-antibody interactions by surfaceplasmon resonance. Applications to epitope mapping.” J. Mol. Recog. 3:208-214. (1990).

Table I lists affinity measurements for representative anti-IGF-IRantibodies of the present invention:

TABLE I Monoclonal K_(d) (M) K_(d) (M) Antibody Protocol 1 Protocol 22.12.1 7.37 × 10⁻⁹ 2.13.2  3.5 × 10⁻⁹ 1.53 × 10⁻⁹ 2.14.3 6.41 × 10⁻¹⁰3.1.1 1.15 × 10⁻⁹ 4.9.2 6.84 × 10⁻¹⁰ 4.27 × 10⁻¹⁰ 4.17.3  1.3 × 10⁻⁸6.1.1 5.65 × 10⁻¹⁰

The kinetic analyses indicates that the antibodies prepared inaccordance with the invention possess high affinities and strong bindingconstants for the extracellular domain of IGF-IR.

Example III Antibody-mediated Inhibition of IGF-I-InducedPhosphorylation of IGF-IR

We performed ELISA experiments in order to determine whether theantibodies of this invention were able to block IGF-I-mediatedactivation of IGF-IR. IGF-I-mediated activation of IGF-IR was detectedby increased receptor-associated tyrosine phosphorylation.

ELISA Plate Preparation

We prepared ELISA capture plates by adding 100 μl blocking buffer (3%bovine serum albumin [BSA] in Tris-buffered saline [TBS]) to each wellof a ReactiBind Protein G-coated 96-well plates (Pierce) and incubatedthe plates with shaking for 30 minutes at room temperature. We dilutedrabbit pan-specific SC-713 anti-IGF-IR antibody (Santa Cruz) in blockingbuffer to a concentration of 5 μg/ml and added 100 μl diluted antibodyto each well. We incubated the plates with shaking for 60-90 minutes atroom temperature. We then washed the plates five times with wash buffer(TBS+0.1% Tween 20) and gently blotted the remaining buffer out ontopaper towels. These plates were not allowed to dry out prior to theaddition of lysate.

Preparation of Lysate from IGF-IR-Expressing Cells

We placed IGF-IR-transfected NIH-3T3 cells (5×10⁴/ml) in 100 μl ofgrowth media (DMEM high glucose media supplemented with L-glutamine(0.29 mg/ml), 10% heat-inactivated FBS, and 500 μg/ml each of geneticin,penicillin and streptomycin) in 96-well U-bottom plates. We incubatedthe plates at 37° C., 5% CO₂ overnight to allow the cells to attach. Wedecanted the media from the plates and replaced it with 100 μl freshgrowth media per well. For testing, we diluted the potential anti-IGF-IRantibodies to five times the desired final concentration in growth mediaand added 25 μl per well. All samples were performed in triplicate. Wethen incubated the plates at 37° C. for one hour. We stimulated thecells with 25 μl/well of 600 μg/ml IGF-1 (prepared in growth media) andincubate the plates at room temperature for 10 minutes. We then decantedthe media by inverting the plates and blotting gently onto paper towelsand lysed the adherent cells by adding 50 μl of lysis buffer (50 mMHEPES, pH 7.4, 10 mM EDTA, 150 mM NaCl, 1.5 mM MgCl₂, 1.6 mM NaVO₄, 1%Triton X-100, 1% glycerol supplemented immediately before use with oneEDTA-free protease inhibitor tablet [Roche Molecular Sciences] per 50ml) and shaking for 5 minutes at room temperature. We added 200 μldilution buffer (50 mM HEPES, pH 7.4, 1.6 mM NaVO₄) to each well andmixed by pipetting up and down. We transferred 100 μl of lysate fromeach well to each well of the ELISA capture plate prepared as describedabove and incubated with gentle shaking for two hours at roomtemperature.

ELISA with Anti-Tyrosine-Phosphate (pTYR) Antibodies

We removed the cell lysate by inverting the plates, washed the platesfive times with wash buffer and blotted on paper towels. We added 100 μlper well pTYR-specific antibody (HRP-PY54) diluted in blocking buffer toa concentration of 0.2 μg/ml and incubated the plates with shaking for30 minutes at room temperature. We then washed these plates five timeswith wash buffer and blotted on paper towels.

We detected binding of the HRP-PY54 antibody by adding 100 μl per wellof TMB peroxidase substrate solution (Kirkegaard & Perry) and incubatingwith shaking as the color developed (approximately 2-10 minutes). Westopped the color development reaction by adding 100 μl per well of TMBstop solution (Kirkegaard & Perry). We then shook the plates for 10seconds at room temperature to mix the solution and quantitated bymeasurement at OD_(450nm).

Table II and FIG. 4 show the results of this experiment performed withseveral antibodies of the invention. The results of this experimentdemonstrate the ability of the antibodies of this invention to blockIGF-I-mediated activation of IGF-IR as shown by increasedreceptor-associated tyrosine phosphorylation. Furthermore, these resultscan be used to quantify the relative potency of the antibodies of thisinvention.

TABLE II Monoclonal IC₅₀ Antibody (μg/ml) 2.12.1 0.172 2.13.2 0.08122.14.3 0.325 4.9.2 0.0324

Example IV Antibody-Mediated Blocking of IGF-I/IGF-IR Binding

antibodies of the invention to inhibit IGF-I binding to IGF-IR in acell-based assay. We plated IGF-IR-transfected NIH-3T3 cells (5×10⁴/ml)in 100 μl of DMEM high glucose media supplemented with L-glutamine (0.29mg/ml), 10% heat-inactivated FBS, and 500 μg/ml each of geneticin,penicillin and streptomycin in 96-well U-bottom plates. We thenincubated the plates at 37° C., 5% CO₂ overnight to allow cells toattach. We then decanted the media from the plates and replaced it with100 μl fresh media per well. For testing, we diluted antibodies in assaymedia (DMEM high glucose media supplemented with L-glutamine, 10%heat-inactivated FBS, 200 μg/ml BSA and 500 μg/ml each of geneticin,penicillin and streptomycin) to the desired final concentration andadded 50 μl per well. All samples were performed in triplicate. We thenincubated the plates at 37° C. for ten minutes. We diluted [¹²⁵I]-IGF-Ito a concentration of 1 μCi/ml is assay media and added 50 μl per wellof the plate. As a control for background radioactivity, we added coldIGF-I to a final concentration of 100 ng/ml. We incubated the plates for10 minutes at 37° C., decanted the media by blotting gently onto papertowels and washed twice with assay media. We then lysed the cells byadding 50 μl 0.1 N NaOH, 0.1% SDS and shaking the plates for fiveminutes at room temperature. We then transferred the samples to ascintillation plate, added 150 μl OptiPhase Supermix and read the signalon a Wallac Micro-Beta counter.

Table III and FIG. 3 show the results of this experiment performed withthree representative antibodies of the invention. This experimentdemonstrated that antibodies of the invention specifically inhibitbinding of [¹²⁵I]-IGF-I to cells overexpressing IGF-IR.

TABLE III Monoclonal Antibody IC₅₀ 2.12.1 0.45 μg/ml 2.13.2 0.18 μg/ml4.9.2  0.1 μg/ml

Example V Epitope Mapping Studies

Having demonstrated that the antibodies of the invention recognizeIGF-IR, we performed epitope mapping studies with several antibodies ofthe invention. We focused these experiments particularly on the 2.12.1,2.13.2, 2.14.3, and 4.9.2 antibodies.

We conducted BIAcore competition studies to determine whether theantibodies of this invention bind to the same or distinct site on theIGF-IR molecule. We bound the extracellular domain (ECD) of IGF-IR to aBIAcore sensorchip as described above in Example II. We bound a firstantibody of the invention to this sensorchip-bound IGF-IR undersaturating conditions. We then measured the ability of subsequentsecondary antibodies of the invention to compete with the primaryantibody for binding to IGF-IR. This technique enabled us to assign theantibodies of this invention to different binding groups.

We performed this experiment with antibodies 2.12.1, 2.13.2, 2.14.3, and4.9.2. We observed that 2.13.2 and 4.9.2 compete for the same site onthe extracellular domain of IGF-IR. The other antibodies, 2.12.1 and2.14.3, bind to sites on IGF-IR that are different from both each otherand from the site bound by 2.13.2 and 4.9.2.

Example VI Species Crossreactivity of the Antibodies of the Invention

In order to determine the species crossreactivity of the antibodies ofthe invention, we performed several experiments includingimmunoprecipitation, antibody-mediating blocking of IGF-I-inducedreceptor phosphorylation and FACS analysis.

To perform immunoprecipitation experiments, we plated cells in DMEM highglucose media supplemented with L-glutamine (0.29 mg/ml), 10%heat-inactivated FBS, and 500 μg/ml each of geneticin, penicillin andstreptomycin to 50% confluence in T25 flasks. We then added 100 μl of anantibody of the invention in Hank's buffered saline solution (HBSS;Gibco BRL) at a concentration of 1 mg/ml. We incubated the plates for 30minutes at 37° C. in an incubator and then stimulated the cells withIGF-I at 100 ng/ml for 10 minutes at room temperature. We lysed thecells in RIPA buffer (Harlow and Lane, supra) and immunoprecipitatedIGF-IR with 2 μg of pan-specific SC-713 anti-IGF-IR antibody (SantaCruz) plus protein A agarose beads for 1 hour at 4° C. We pelleted thebeads and wash three times with PBS/T (PBS+0.1% Tween-20) and thenboiled the beads in 40 μl Laemmli buffer containing 5% βME.

The samples prepared as described above were then analyzed by Westernblot. We loaded 12 μl of each sample per lane on 4-10% gradient Novex™gels run with 1×MES buffer (Novex™). Gels were run at 150V for 1 hour orat 200V for approximately 30 minutes. We then transferred the gel to amembrane in Novex™ transfer buffer with 10% methanol either overnight at100 mA or for 1-1.5 hours at 250 mA. We then allowed the membrane to drycompletely and blocked at room temperature with TBS (Tris-bufferedsaline pH 8.0) containing Superblock (Pierce Chemical Co.). We added theIGF-IR blotting antibody SC713 (Santa Cruz) to detect immunoprecipitatedIGF-IR.

This experiment was performed with antibodies of the invention,particularly 2.12.1, 2.13.2, 4.17.3 and 4.9.2, on cells from a varietyof animals. We found that antibodies 2.12.1, 2.13.2 and 4.9.2 were ableto bind human, but not canine, guinea pig, rabbit or IGF-IR. Further,these antibodies were able to bind COS7 and Rhesus IGF-IR, both derivedfrom old world monkeys, but not IGF-IR from the marmoset, which is a newworld monkey. These experiments indicate that the antibodies are highlyspecific.

Antibody-Mediated Blocking of IGF-I/IGF-IR Binding in Non-Human Primates

Following our observation that the antibodies of the invention recognizeIGF-IR from old world monkeys, we also tested their ability to blockIGF-I/IGF-IR binding in cells derived from these old world monkeys. Weplated cells in DMEM high glucose media supplemented with L-glutamine,10% heat-inactivated FBS, and 500 μg/ml each of geneticin, penicillinand streptomycin to 50% confluence in T25 flasks. We then added anantibody of the invention, or media without antibody as a control, andstimulated the cells with IGF-I at 100 ng/ml for 10 minutes at roomtemperature. After stimulation, we lysed the cells andimmunoprecipitated IGF-IR with pan-specific IGF-IR antibody SC713 asdescribed above. We then performed Western blot analysis as describedabove using HRP-PY54 antibody to detect phosphorylated tyrosine in theactivated IGF-IR.

We observed that antibodies of this invention, in particular 2.13.2 and4.9.2 could block IGF-I-induced phosphorylation of IGF-IR in both COS7and Rhesus cells. The IC₅₀ for the observed inhibition was 0.02 μg/mland 0.005 μg/ml for COS7 and Rhesus IGF-IR, respectively.

Determination of Cross-species Affinity of Antibodies of the Invention

We performed FACS analysis to determine the affinity of the antibodiesof the invention for IGF-IR from other animals, particularly the oldworld monkeys described above. We incubated aliquots of human and monkeycells (5×10⁵) for 1 hour on ice with increasing concentrations ofbiotinylated anti-IGF-IR antibodies of the invention or with abiotinylated anti-keyhole limpet hemocyanin (KLH) antibody (Abgenix) asa negative control. We then incubated the samples for 30 minutes on icewith steptavidin-conjugated RPE (phycoerythrin). We measured binding byflow cytometry and analyzed the histograms of fluorescence intensity(Fl2-H) versus cell number (Counts) using CellQuest software. Wecalculated binding (K_(d)) for each antibody from graphs of meanfluorescence intensity versus antibody concentration. In mostexperiments, we measured binding in cultured human MCF-7 cells andeither rhesus or cynomologous tissue culture cells. We controlled fordepletion of the antibody by measuring binding over a range of cellconcentrations.

We performed the aforementioned FACS analysis to test the ability ofantibodies of the invention, particularly 2.13.2 and 4.9.2, to bindhuman, rhesus and cynomologous cells. We observed a half maximal binding(K_(a)) of 0.1 μg/ml for all cell lines tested.

Example VII IGF-I Receptor Downregulation

We performed blocking experiments essentially as described above inExample IV up to the addition of [¹²⁵I]-labeled IGF-I. At this point, weboiled the cells in 40 μl Laemmli buffer containing 50% βme. We thenanalyzed the samples by western blot analysis as described above inExample VI and probed the blots with both pan-specific IGF-IR antibodySC713 to quantify the levels of IGF-IR and HRP-PY54 antibody to monitorthe levels of phosphorylated tyrosine in the activated IGF-IR.

As observed previously (Example III), we observed blockage ofIGF-1-induced phosphorylation of IGF-IR following the treatment of cellswith an antibody of this invention (FIG. 4). Further, we observed thatthis blockage of IGF-I-induced phosphorylation was followed bydownregulation of the IGF-IR in these cells. See, e.g., FIG. 4. IGF-IRlevels were maximally reduced 16 hours after stimulation with IGF-I inthe presence of an antibody of the invention.

Example VIII Effects of the Antibodies of the Invention on IGF-IR InVivo

We determined whether the effects of the antibodies of the invention onIGF-IR as described in the previous examples would occur in vivo. Weinduced tumors in athymic mice according to published methods (V. A.Pollack et al., “Inhibition of epidermal growth factorreceptor-associated tyrosine phosphorylation in human carcinomas withCP-358,774: Dynamics of receptor inhibition in situ and antitumoreffects in athymic mice,” J. Pharmacol. Exp. Ther. 291:739-748 (1999).Briefly, we injected IGF-IR-transfected NIH-3T3 cells (5×10⁶)subcutaneously into 3-4 week-old athymic (nu/nu) mice with 0.2 ml ofMatrigel preparation. We then injected mice with an antibody of theinvention intraperitoneally after established (i.e. approximately 400mm³) tumors formed.

After 24 hours, we extracted the tumors, homogenized them and determinedthe level of IGF-IR. To determine IGF-IR levels, we diluted the SC-713antibody in Blocking buffer to a final concentration of μg/ml and added100 μl to each well of a Reacti-Bind Goat anti-rabbit (GAR) coated plate(Pierce). We incubated the plates at room temperature for 1 hour withshaking and then washed the plates five times with wash buffer. We thenweighed tumor samples that had been prepared as described above andhomogenized them in lysis buffer (1 ml/100 mg). We diluted 12.5 μl oftumor extract with lysis buffer to a final volume of 100 μl and addedthis to each well of a 96-well plate. We incubated the plates at roomtemperature with shaking for 1-2 hours and then washed the plates fivetimes with Wash buffer. We then added 100 μl HRP-PY54 or biotinylatedanti-IGF-IR antibody in Blocking buffer to each well and incubated atroom temperature with shaking for 30 minutes. We then washed the platesfive times with wash buffer and developed the plates. We developed theplates probed with HRP-PY54 by adding 100 μl of the TMB microwellsubstrate per well and stopped color development with the addition 100μl 0.9 M H₂SO₄. We then quantitated the signal by shaking for 10 secondsand measuring OD_(450nm). The signal was normalized to total protein. Wedeveloped plates probed with anti-IGF-IR antibody by adding 100 μl ofstreptavidin-HRP diluted in Blocking buffer to each well, incubating atroom temperature with shaking for 30 minutes and then continuing asdescribed for HRP-PY54.

We observed that intraperitoneal injection of an antibody of thisinvention, particularly 2.13.2 and 4.9.2, resulted in inhibition ofIGF-IR activity as measured by a decrease of both IGF-IR phosphotyrosine(phosphorylated IGF-IR) and total IGF-IR protein (FIG. 6). In addition,we also observed a decrease in IGF-IR phosphotyrosine (phosphorylatedIGF-IR) (FIG. 5). Without wishing to be bound by any theory, thedecreased levels of IGF-IR phosphotyrosine may be due to the decreasedlevels of IGF-IR protein in vivo after treatment with the antibody ormay be due to a combination of decreased levels of IGF-IR protein and adecrease in tyrosine phosphorylation on the IGF-IR that is present dueto blocking of activation by ligand (e.g., IGF-I or IGF-II).Furthermore, this inhibition was responsive to the dose of antibodyinjected (FIG. 6). These data demonstrate that the antibodies of theinvention are able to target the IGF-IR in vivo in a manner analogous towhat we observed in vitro.

Example IX Growth Inhibition (TGI) of 3T3/IGF-IR Cell Tumors

We tested whether anti-IGF-IR antibodies of the invention would functionto inhibit tumor growth. We induced tumors as described above (ExampleVIII) and when established, palpable tumors formed (i.e. 250 mm³, within6-9 days), we treated the mice with a single, 0.20 ml dose of antibodyby intraperitoneal injection. We measured tumor size by Vernier calipersacross two diameters every third day and calculated the volume using theformula (length×[width]²)/2 using methods established by Geran, et al.,“Protocols for screening chemical agents and natural products againstanimal tumors and other biological systems,” Cancer Chemother. Rep.3:1-104.

When we performed this analysis with an antibody of the invention, wefound that a single treatment with antibody 2.13.2 alone inhibited thegrowth of IGF-IR-transfected NIH-3T3 cell-induced tumors (FIG. 7, leftpanel). Furthermore, in combination studies with a single dose of 7.5mg/kg intravenously-supplied adriamycin, we observed that administrationof a single dose of 2.13.2 enhanced the effectiveness of adriamycin, aknown inhibitor of tumor growth. The combination of adriamycin with anantibody of the invention, 2.13.2, demonstrated a growth delay of 7 daysversus treatment with the antibody or adriamycin alone (FIG. 7, rightpanel).

Example X Relationship of Antibody Levels to IGF-IR Downregulation

Tumors were induced in nude mice as described in Example VIII. The micewere then treated with 125 μg of 2.13.2 by intraperitoneal injuction, asdescribed in Example VIII. Tumors were extracted and IGF-IR levels weremeasured by ELISA as described in Example VIII. FIG. 8 shows the serum2.13.2 antibody levels and IGF-IR receptor levels over time. Theexperiment demonstrates that the IGF-IR is down-regulated by theantibody and that the degree of IGF-IR inhibition is dose proportionalto the serum concentration of the antibody.

Example XI Growth Inhibition of 3T3/IGF-IR Tumors with Multiple Dosingof Antibody in Combination with Adriamycin

Tumors were induced in nude mice as described in Example IX. Mice withestablished subcutaneous tumors of approximately 250 mm³ were treated ondays 1, 8, 15 and 22 with various amounts of 2.13.2 antibody (i.p.) or7.5 mg/kg adriamycin (i.v.), either as single agents or in combination,as described in Example IX. FIG. 9 shows the tumor size in relation tothe various treatments over time. The experiment demonstrates thattreatment with an anti-IGF-IR antibody given once every seven daysinhibits tumor cell growth and enhances inhibition of tumor cell growthin combination with adriamycin, a known tumor inhibitor.

Example XII Growth Inhibition of Large Tumors

Tumors were induced in nude mice as described in Example IX. Mice withlarge established subcutaneous tumors of slightly less than 2000 mm³were treated on days 1 and 8 with various amounts of 2.13.2 antibody(i.p.) or 7.5 mg/kg adriamycin (i.v.), either as single agents or incombination, as described in Example IX. FIG. 10 shows the tumor size inrelation to the various treatments over time. Control, antibody aloneand adriamycin alone animal groups were terminated at day 5, when thetumor size exceeded 2000 mm³. The experiment demonstrates that treatmentwith an anti-IGF-IR antibody in combination with adriamycin is highlyefficacious against large tumors when multiple doses are given.

Example XIII Growth Inhibition of Colorectal Cell Tumors

Tumors were induced in nude mice as described in Example IX except thatColo 205 cells (ATCC CCL 222) were used. Colo 205 cells are humancolorectal adenocarcinoma cells. Mice with established subcutaneoustumors of approximately 250 mm³ were treated with various amounts of2.13.2 antibody (i.p.) or with 100 mg/kg 5-fluorodeoxyuridine (5-FU,i.v.), either as single agents or in combination, as described inExample IX. FIG. 11 shows the tumor size in relation to the varioustreatments over time. The experiment demonstrates that treatment with ananti-IGF-IR antibody given once inhibits human colorectal cancer cellgrowth when provided as a single agent and enhances the effectiveness of5-FU, a known tumor inhibitor. Mice with established Colo 205 tumorswere treated on days 1, 8, 15 and 22 with 500 μg 2.13.2 (i.p.), 100mg/kg 5-FU (i.v.) or a combination thereof. FIG. 12 shows the tumor sizein relation to the various treatments over time. The experimentdemonstrates that treatment with an anti-IGF-IR antibody given onceevery seven days inhibits human colorectal cancer cell growth andenhances the effectiveness of 5-FU.

Example XIV Growth Inhibition of Breast Cancer Cell Tumors

Nude mice as described in Example VIII were implanted with biodegradableestrogen pellets (0.72 mg 17-

-estradiol/pellet, 60 day release; Innovative Research of America).After 48 hours, tumors were induced in nude mice essentially asdescribed in Example IX except that MCF-7 cells (ATCC HTB-22) were used.MCF-7 cells are estrogen-dependent human breast carcinoma cells. Micewith established subcutaneous tumors of approximately 250 mm³ weretreated with 50:g 2.13.2 antibody (i.p.) on days 1, 4, 7, 10, 13, 16, 19and 22 (q3dx7) or with 6.25 mg/kg taxol (i.p.) on days 1, 2, 3, 4, 5(q1dx5), either as single agents or in combination, essentially asdescribed in Example IX. FIG. 13 shows the tumor size in relation to thevarious treatments over time. The experiment demonstrates that treatmentwith an anti-IGF-IR antibody by itself inhibits human breast cancer cellgrowth when administered once every three days and also enhances theeffectiveness of taxol, a known breast cancer inhibitor, when given incombination.

Mice having established tumors from MCF-7 cells as described immediatelyabove were treated on day 1 with various amounts of 2.13.2 antibody(i.p.) alone or with 3.75 mg/kg adriamycin (i.v.), essentially asdescribed in Example IX. FIG. 14 shows the tumor size in relation to thevarious treatments over time. The experiment demonstrates that a singletreatment with an anti-IGF-IR antibody by itself inhibits human breastcancer cell growth and enhances the effectiveness of adriamycin, a knowntumor inhibitor.

Mice having established tumors from MCF-7 cells as described immediatelyabove were treated with 250:g 2.13.2 antibody (i.p.) on days 1, 8, 15and 23 or with a biodegradable tamoxifen pellet (25 mg/pellet, freebase, 60 day release, Innovative Research of America), either as singleagents or in combination, essentially as described in Example IX. Thetamoxifen pellet was implanted on day 1 after the tumor was established.FIG. 15 shows the tumor size in relation to the various treatments overtime. The experiment demonstrates that treatment with an anti-IGF-IRantibody administered once every seven days inhibits human breast cancergrowth by itself and enhances the effectiveness of tamoxifen, a knowntumor inhibitor.

Example XVI Growth Inhibition of Epidermoid Carcinoma Cell Tumors

Tumors were induced in nude mice essentially as described in Example IXexcept that A431 cells (ATCC CRL 1555) were used. A431 cells are humanepidermoid carcinoma cells that overexpress EGFR. Mice with establishedsubcutaneous tumors of approximately 250 mm³ were treated on days 1, 8,15, 22 and 29 with 500:g 2.13.2 antibody (i.p.) or were treated oncedaily for 27 days with 10 mg/kg CP-358,774 given orally (p.o.), eitheras single agents or in combination, as described in Example IX.CP-358,774 is described in U.S. Pat. No. 5,747,498 and Moyer et al.,Cancer Research 57: 4838-4848 (1997), herein incorporated by reference.FIG. 16 shows the tumor size in relation to the various treatments overtime. The experiment demonstrates that treatment with an anti-IGF-IRantibody enhances the effectiveness of CP-358,774, a known EGF-Rtyrosine kinase inhibitor, for inhibiting the growth of a humanepidermoid carcinoma tumor.

Example XVII Pharmacokinetics of Anti-IGF-IR Antibodies in vivo

To evaluate the pharmacokinetics of the anti-IGF-IR antibodies,cynomolgus monkeys were injected intravenously with 3, 30 or 100 mg/kgof 2.13.2 antibody in an acetate buffer. Serum was collected from themonkeys at various time points and anti-IGF-IR antibody concentrationsin the monkeys were determined for a period of up to ten weeks levels.To quantitate functional serum antibody levels, the extracellular domainof the human IGF-IR (IGF-I-sR, R&D Systems, Catalog #391GR) was bound to96-well plates. Monkey serum (diluted between 1:100 and 1:15,000) wasadded to the assay plates so that each sample would be within the linearrange of the standard curve and incubated under conditions in which anyanti-IGF-IR antibody would bind to IGF-I-sR. After washing the plates, alabeled anti-human IgG antibody was added to the plates and incubatedunder conditions in which the anti-human IgG antibody would bind to theanti-IGF-IR antibody. The plates were then washed and developed, and acontrol standard curve and linear regression fits used to quantitate theamount of anti-IGF-IR antibodies. FIG. 17 shows the concentration of2.13.2 in serum over time. The experiment demonstrates that thehalf-life of the anti-IGF-IR antibody is 4.6 to 7.7 days and has avolume distribution of 74-105 mL/kg. Further, the experimentdemonstrates that the amounts given are dose-proportional in the monkey,which indicates that the anti-IGF-IR antibody has saturated anyavailable IGF-IR binding sites in the body even at the lowest dose of 3mg/kg.

Example XVIII Combination Therapy of Anti-IGF-IR Antibody and AdriamycinDownregulates IGF-IR In Vivo

Tumors were induced in nude mice as described in Example IX. Mice withestablished subcutaneous tumors of approximately 400 mm³ were treatedwith a single injection of 250:g 2.13.2 antibody (i.p.) or with 7.5mg/kg adriamycin (i.v.), either as single agents or in combination, asdescribed in Example IX. 72 hours after administration of the agents,tumors were extracted as described in Example VIII, and equal amounts ofthe tumor extracts were subjected to sodium dodecyl phosphatepolyacrylamide gel electrophoresis (SDS PAGE) and western blot analysisusing the anti-IGF-IR antibody SC-713 (Santa Cruz). FIG. 18 shows theamounts of IGF-IR in tumor cells in control animals (first three lanesof each panel), in animals treated with antibody alone (top panel), inanimals treated with adriamycin alone (middle panel) and in animalstreated with antibody and adriamycin (lower panel). Each lane representsequal amounts of protein from individual tumors from individual mice.The experiment demonstrates that treatment with adriamycin alone haslittle effect on IGF-IR levels and that treatment with antibody aloneshows some decrease in IGF-IR levels. Surprisingly, treatment withadriamycin and antibody together shows a dramatic decrease in IGF-IRlevels, demonstrating that adriamycin and antibody greatly downregulateIGF-IR levels.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

Applicants also incorporate by reference herein the document“0006590032106.txt,” which was created on Dec. 17, 2013 and has a sizeof 65,861 bytes, and which is electronically submitted concurrently withthis application.

We claim:
 1. A humanized, chimeric or human monoclonal antibody orantigen-binding portion thereof that specifically binds to insulin-likegrowth factor I receptor (IGF-IR).
 2. The antibody or portion thereofaccording to claim 1, wherein the IGF-IR is human.
 3. The antibody orantigen-binding portion thereof according to claim 1, wherein theantibody or portion thereof has at least one property selected from thegroup consisting of: a) does not bind to mouse, rat, dog or rabbitIGF-IR; b) binds to cynomologous or rhesus IGF-IR but not to marmosetIGF-IR; c) inhibits the binding of IGF-I or IGF-II to IGF-IR. d) has aselectivity for IGF-IR that is at least 50 times greater than itsselectivity for insulin receptor; e) inhibits tumor growth in vivo; f)causes IGF-IR disappearance from the cell surface when incubated with acell expressing IGF-IR; g) inhibits IGF-IR-induced tyrosinephosphorylation; h) binds to IGF-IR with a K_(d) of 8×10⁻⁹ M or less;and i) has an off rate for IGF-IR of K_(off) of 10⁻⁴ or smaller.
 4. Theantibody or antigen-binding portion thereof according to claim 3,wherein the antibody or portion thereof has all of said properties. 5.The antibody or antigen-binding portion thereof according to claim 1,wherein the antibody or portion thereof has at least one propertyselected from the group consisting of: a) cross-competes for binding toIGF-IR with an antibody selected from the group consisting of 2.12.1,2.13.2, 2.14.3, 3.1.1, 4.9.2 and 4.17.3; b) binds to the same epitope ofIGF-IR as an antibody selected from the group consisting of 2.12.1,2.13.2, 2.14.3, 3.1.1, 4.9.2 and 4.17.3; c) binds to the same antigen asthat bound by the antibody selected from the group consisting of 2.12.1,2.13.2, 2.14.3, 3.1.1, 4.9.2 and 4.17.3; d) binds to IGF-IR withsubstantially the same K_(d) as an antibody selected from the groupconsisting of 2.12.1, 2.13.2, 2.14.3, 3.1.1, 4.9.2 and 4.17.3; and e)binds to IGF-IR with substantially the same off rate as an antibodyselected from the group consisting of 2.12.1, 2.13.2, 2.14.3, 3.1.1,4.9.2 and 4.17.3.
 6. The antibody or antigen-binding portion thereofaccording to claim 5, wherein the antibody or portion thereof comprisesall of said properties.
 7. The antibody or antigen-binding portionthereof according to claim 1, wherein said antibody or portion thereofinhibits binding between IGF-IR and IGF-I or IGF-II with an IC₅₀ of lessthan 100 nM.
 8. The antibody or antigen-binding portion thereofaccording to claim 1, wherein said antibody or antigen-binding portionthereof comprises a variable region of a κ light chain, wherein thesequence of said variable region of said κ light chain comprises no morethan ten amino acid changes from the amino acid sequence encoded by agermline Vκ A30, A27 or O12 gene.
 9. The antibody or antigen-bindingportion thereof according to claim 1, wherein said antibody orantigen-binding portion thereof comprises a variable region of a heavychain, wherein the sequence of said variable region comprises no morethan eight amino acid changes from the amino acid sequence encoded by agermline V_(H) DP47, DP35, DP71 or VIV-4 gene.
 10. A pharmaceuticalcomposition comprising the antibody or portion thereof according toclaim 1 and a pharmaceutically acceptable carrier.
 11. A process formaking an antibody that binds to IGF-IR, comprising the steps of: a)immunizing a non-human mammal with an immunogen comprising IGF-IR,wherein the mammal is capable of expressing human antibodies in B cellsof the animal; b) isolating B cells from the mammal; c) screening said Bcells, or cell lines derived therefrom, to identify a cell line thatproduces antibodies that bind to IGF-IR; d) culturing the cell line thatexpresses antibodies that bind to IGF-IR; and e) isolating antibodiesthat bind to IGF-IR from the cell line.
 12. An isolated cell line thatproduces the antibody according to claim
 1. 13. A method of diagnosingthe presence or location of an IGF-IR-expressing tumor in a subject inneed thereof, comprising the steps of a) injecting the antibodyaccording to claim 1 into the subject, b) determining the expression ofIGF-IR in the subject by localizing where the antibody has bound, c)comparing the expression in part (b) with that of a normal referencesubject or standard, and d) diagnosing the presence or location of thetumor.
 14. A method of treating a patient in need thereof with theantibody or antigen-binding portion thereof according to claim 1,comprising the step of administering to the patient an effective amountof the antibody.
 15. An isolated nucleic acid molecule that comprises anucleic acid sequence that encodes a heavy chain or antigen-bindingportion thereof or a light chain or antigen-binding portion thereof ofan antibody according to claim
 1. 16. A vector comprising the nucleicacid molecule according to claim 15, wherein the vector optionallycomprises an expression control sequence operably linked to the nucleicacid molecule.
 17. A host cell comprising the nucleic acid moleculeaccording to claim
 15. 18. A method of making an anti-IGF-IR antibody orantigen-binding portion thereof, comprising culturing the host cellaccording to claim 17 under suitable conditions and recovering saidantibody or antigen-binding portion.
 19. A non-human transgenic animalcomprising the nucleic acid according to claim 15, wherein the non-humantransgenic animal expresses said nucleic acid.
 20. A method of treatinga subject in need thereof with an antibody or antigen-binding portionthereof that specifically binds to IGF-IR, comprising the steps of (a)administering an effective amount of an isolated nucleic acid moleculeencoding the heavy chain or the antigen-binding portion thereof, anisolated nucleic acid molecule encoding the light chain or theantigen-binding portion thereof, or both the nucleic acid moleculesencoding the light chain and the heavy chain or antigen-binding portionsthereof; and (b) expressing the nucleic acid molecule.